Correlation of Moraxella catarrhalis macrolide susceptibility with the ability to adhere and invade human respiratory epithelial cells.

Recently, the prevalence of macrolide-resistant Moraxella catarrhalis has been reported, especially among Chinese children. The fitness cost of resistance is reported to render the resistant bacteria less virulent. To investigate the correlation between macrolide susceptibility of M. catarrhalis and pathogenicity, the whole genome of 70 M. catarrhalis isolates belonging to four clonal complexes with different macrolide susceptibilities was sequenced. The gene products were annotated with the Gene Ontology terms. Based on 46 extracted essential virulence genes, 19 representative isolates were selected to infect type II alveolar cells (A549 cells). The ability of these isolates to adhere and invade human epithelial cells and to produce cytokines was comparatively analysed. Furthermore, mice were infected with a pair of M. catarrhalis isolates with different pathogenic behaviours and macrolide susceptibilities to examine pulmonary clearance, histological findings, and the production of cytokines. The percentages of annotations for binding, metabolic process, cellular process, and cell were non-significantly different between the macrolide-resistant and macrolide-susceptible groups. The presence of uspA2, uspA2H, pilO, lbpB, lex1, modM, mboIA, and mboIB significantly differed among the four clonal complexes and macrolide susceptibility groups. Furthermore, compared with those in macrolide-susceptible isolates, the adhesion ability was stronger (P = 0.0019) and the invasion ability was weaker (P < 0.0001) in the macrolide-resistant isolates. Mouse experiments revealed that pulmonary macrophages elicit immune responses against M. catarrhalis infection by significantly upregulating the Csf2, Il4, Il13, Il1b, Il6, Tnf, and Il18. Therefore, M. catarrhalis populations exhibited diverse pathogenicity in vitro and in vivo.

Landscape of PD-1/PD-L1 Regulation and Targeted Immunotherapy.

Programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) is a significant immune checkpoint, and the dysfunction of this axis contributes to tumor metastasis and immune escape. PI3K/Akt/mTOR and MAPK signal network induces PD-1/PD-L1 expression and facilitates tumor progression. Transcriptional factors such as hypoxia induced factors, PTEN, p53, CDK5, BRD4, STAT modulate PD-1/PD-L1 expression. PD-1/PD-L1 level is also regulated via epigenetic and post-translational manner. The underlying mechanisms mentioned above may provide potential targets for tumor treatment. At present, the combination therapy of PD-1/PD-L1 monoclonal antibodies plus small molecular inhibitors has achieved good outcomes in tumor treatment.

[Analysis of cytomegaloviral antigenemia test and re-test after antiviral chemotherapy in patients with autoimmune and non-autoimmune diseases].

OBJECTIVE: To retrospectively analyze the cytomegalovirus (CMV) antigenemia test and re-test after antiviral chemotherapy in patients with autoimmune and non-autoimmune diseases. METHODS: CMV Brite kit and indirect immunofluorescence were used to detect CMVpp65 antigenemia in 6471 peripheral blood leukocyte specimens from 5325 clinic and hospitalized patients with clinically suspicious CMV infections from May 2008 to February 2012. And the positive results were defined as episodes of systemic CMV activity. RESULTS: In 6471 EDTA-treated peripheral blood specimens, 948 (14.6%) were found with positive CMV antigenemia. The average positive rate from 13 kinds of autoimmune diseases was 34.9% (670/1922) in which systemic lupus erythematosus patients had the highest (52.4%, 551/1052). Meanwhile, the average positive rate from 12 kinds of non-autoimmune diseases was only 6.1% (144/2367) in which it was 17.3% (27/156) in patients with respiratory/acute renal failure, acquired immunodeficiency syndrome (AIDS) and kidney transplant recipients. And 189 patients with positive antigenemia were re-tested after antiviral chemotherapy and only 64 (33.9%) were converted negatively. CONCLUSIONS: Patients with autoimmune diseases have replaced traditionally immunocompromised patients, e.g. AIDS and kidney transplant recipient, to become the highest risk group of systemic CMV activity. Negative conversion rate of CMV antigenemia is low after antiviral chemotherapy.

Construction of a multiple myeloma diagnostic model by magnetic bead-based MALDI-TOF mass spectrometry of serum and pattern recognition software.

A diagnosis of multiple myeloma (MM) is difficult to make on the basis of any single laboratory test result. Accurate diagnosis of MM generally results from a number of costly and invasive laboratory tests and medical procedures. The aim of this work is to find a new, highly specific and sensitive method for MM diagnosis. Serum samples were tested in groups representing MM (n = 54) and non-MM (n = 108). These included a subgroup of 17 plasma cell dyscrasias, a subgroup of 17 reactive plasmacytosis, 5 B cell lymphomas, and 7 other tumors with osseus metastasis, as well as 62 healthy donors as controls. Bioinformatic calculations associated with MM were performed. The decision algorithm, with a panel of three biomarkers, correctly identified 24 of 24 (100%) MM samples and 46 of 49 (93.88%) non-MM samples in the training set. During the masked test for the discriminatory model, 26 of 30 MM patients (sensitivity, 86.67%) were precisely recognized, and all 34 normal donors were successfully classified; patients with reactive plasmacytosis were also correctly classified into the non-MM group, and 11 of the other patients were incorrectly classified as MM. The results suggested that proteomic fingerprint technology combining magnetic beads with MALDI-TOF-MS has the potential for identifying individuals with MM. The biomarker classification model was suitable for preliminary assessment of MM and could potentially serve as a useful tool for MM diagnosis and differentiation diagnosis.

[Analysis of the correlation between PreS1-Ag, PreS2-Ag, HBeAg and HBV DNA in the serum of chronic hepatitis B patients].

OBJECTIVE: To investigate the correlation between the serum indices and HBV DNA. METHODS: 100 chronic HBV patients and 40 healthy controls were enrolled in this study. The patients have not received any treatment with interferon (IFN) and similar nucleotide, and featured with normal ALT/AST, positive HBV DNA examined by using internal-quantitative standard PCR. Serum indices were measured by micro-particle enzyme immunoassay analysis and ELISA. RESULTS: In chronic HBV patients with positive HBV DNA, the positive percentage were 75% for HBeAg, 25% for anti-HBe, 69% for PreS1-Ag, and 77% for PreS2-Ag. In 75 HBeAg positive cases, there were 54 cases of PreS1-Ag positive and 60 cases of PreS2-Ag positive; while among anti-HBe positive cases, there were 14 cases of PreS1-Ag positive and 17 cases of PreS2-Ag positive. There was no significant difference of the positive percentage of PreS1-Ag and PreS2-Ag between HBeAg positive and anti-HBe positive (both P > 0.05). Under circumstance of HBeAg positive, the percentage of both PreS1-Ag and PreS2-Ag negative (8%) was significant lower than that of anti-HBe positive (28%) (P < 0.05). There were 72 cases with consistent positive or negative for both PreS1-Ag and PreS2-Ag. CONCLUSION: For hepatitis B patients with positive HBV DNA, the differences of positive percentages for HBeAg, PreS1-Ag and PreS2-Ag are moderate. The positive percentages of PreS1-Ag and PreS2-Ag do not correlate with HBeAg positive. The negative percentages of PreS1-Ag and PreS2-Ag correlate with anti-HBe positive.

[A modified dye test for Toxoplasma gondii infection].

A modified dye test with microplate was to be established to detect Toxoplasma antibodies with cell-cultured Toxoplasma gondii. Numbers of stained and unstained tachyzoites were estimated in every 100 tachyzoites in each well after dyeing with methylene blue. The dilution with 50% tachyzoites stained was used as final dilution. Better results of the microplate dye test has been received when the concentration of tachyzoites in suspension reaches 10(9)/ml with 1% sodium citrate as accessory factor.

[Study on the correlation among quantification of HBV-DNA and HBeAg, anti-HBe in hepatitis B carriers].

OBJECTIVE: To better understand the duplication of hepatitis B virus (HBV) in order to improve clinical diagnoses and treatments via quantitative measurement of HBV-DNA and comparison of correlation of HBV-DNA with HBeAg and anti-HBe. METHODS: For 883 hepatitis B patients with positive HBsAg, HBV-DNA was measured by COBAS AMPLICOR HBV MONITOR reagent and COBAS AMPLICOR quantitative PCR instrument. Microparticle enzyme immunoassay analysis (MEIA) was then carried out with fully automatic enzyme immunoassay analysis instrument made by Abbott Axsym from the U.S. to measure HBeAg and anti-HBe. Correlation was analysed by SPSS. RESULTS: (1)Positive correlation between 690 HBV-DNA positive and HBeAg positive with r= 0. 505 (P< 0.01) was found with mean values as:HBV-DNA:7.12 x 10(12) copies/ml;HBeAg:218.31 S/CO. HBV-DNA:10(4) copies/ml, HBeAg: 104 S/CO; HBV-DNA: 10(5)-10(8) copies/ml, HBeAg: 112 S/CO; HBV-DNA: 10(9)-10(15) copies/ml, HBeAg: 252 S/CO. (2) No correlation was found between 193 HBV-DNA and anti-HBe + with r= -0.052(P= 0.477> 0.05) with Mean: HBV-DNA: 8.0x 10(10) copies/ml anti-HBe: 0.18 S/CO. CONCLUSION: HBV-DNA and HBeAg appeared to have had linear correlation, showing that HBeAg> 100 S/CO,HBV-DNA> 10(4) copies/ml and hepatitis B virus were reproduced. However, HBV-DNA did not show linear correlation with anti-HBe as HBeAg negative and anti-HBe positive, the level of hepatitis B viral replication decrease slightly. But the virus load is still high. Infectivity can not neglect.

[Correlation of hepatitis B e antigen with hepatitis B virus DNA in the serum of chronic hepatitis B patients after treatment].

OBJECTIVE: To analyze the correlation of hepatitis B virus (HBV) DNA with the serological markers of HB in the serum of chronic HB patients after treatment PCR method and to analyze the status of these markers and the multiplication of virus. METHODS: Peripheral blood samples were collected from 480 chronic HB patients, aged 15 - 50, who had been treated by anti-nucleotide drugs or traditional Chinese herbs and showed normal ALT/AST. Both COBAS AMPLICOR HBV MONIORTM kit (internal-standard PCR method) and Light Cycler real time fluorescent quantitative PCR instrument (external-quantitative standard PCR method) were used to measure the HBV DNAS level. 42 of the 312 patients with the HBV DNA level lower than the minimum test limit measured by COBAS AMPLICOR HBV MONIORTM kit and HBeAg positive (>4 S/CO) underwent microparticle enzyme immunoassay (MEIA) to test the HBsAg, anti-HBs, HBeAg, anti-HBe, and HBcIgG. RESULTS: Seven of the 42 patients with HBV DNA negative measured by COBAS AMPLICOR HBV MONIORTM kit lower then the minimum test limit were shown as HBV DNA positive by Light Cycler real time fluorescent quantitative PCR. The 42 patients were HBsAg (+), anti-HBs (-), HBeAg (+), anti-HBe (-), and anti-HBcIgG (-), with an average HBeAg level of 42.26 S/CO and a positive HBeAg rate of 13.46%. CONCLUSION: HBeAg positivity does not necessarily means an active multiplication of HBV. The changes of the serological markers of HBV may be not consistent with that of HBV DNA. It is more objective to undergo both internal-standard and external-quantitative standard methods.

[Animal model of facial neuritis induced by herpes simplex virus].

OBJECTIVE: To study the role of herpes simplex virus type 1 ( HSV-1 ) in facial paralysis by developing an experimental animal model of viral facial paralysis. METHODS: Both sides of posterior auricular branch of facial nerve were anatomies and incised in 66 mice. The HSV-1 was inoculated into right ear branch and fetal bovine serum was inoculated into left ear branch as control. The symmetry of mouse face was observed and scored. The temporal bones were serially sectioned and stained with hematoxylin and eosin. The extratemporal facial nerves were stained with osmium tetroxide. HSV-1 DNA in bilateral facial nerve, brain stem, trigeminal ganglion and spinal cord was detected by the polymerase chain reaction. RESULTS: Twenty-eight (42. 42%) mice developed right facial paralysis between 2 and 5 days after inoculation. Continuing 3-6 days, the facial paralysis recovered spontaneously. Thirty-eight mice had no signs of facial paralysis. Compared with the left, nerve swelling, inflammatory cell infiltration were manifested in right temporal facial nerve of paralyzed mice. The ratio of the cross-sectional area of the facial nerve to the facial canal ( FN/FC ) was significantly higher than that on the control side (P < 0.01). Demyelinated nerve fibers were seen in the right extratemporal facial nerve. Not only in paralyzed mice, but also in non-paralyzed mice, HSV DNA was detected in some nerve tissues. CONCLUSIONS: Inoculating HSV-1 into posterior auricular branch of facial nerve can produce an acute and transient facial paralysis in mice. The possible pathophysiologic mechanism of the facial paralysis is viral invasion and transportation from distal branch to main trunk. Then the viral facial neuritis causes facial paralysis.

Role of perioperative parenteral nutrition in severely malnourished patients with Crohn's disease.

AIM: To evaluate the effect of perioperative parenteral nutrition on serum immunoglobulin, weight change, and post-operative outcome in severely malnourished patients with Crohn's disease. METHODS: Thirty-two severely malnourished patients with Crohn's disease who had undergone surgery in our hospital were reviewed. Sixteen patients who received perioperative parenteral nutrition were enrolled in the study group, and the other 16 patients who did not receive parenteral nutrition were enrolled in the control group. Serum immunoglobulin, body mass index (BMI), liver function, weight change, and postoperative complications were evaluated. RESULTS: Serum IgM levels elevated 1 wk before surgery in both groups, and decreased to normal value (from 139+/-41 to 105+/-29 mg/dL, P = 0.04) 4 wk after operation in the study group, while no significant changes was noted in the control group (from 133+/-16 to 129+/-13 mg/dL, P = 0.34). There were no significant changes in concentrations of IgG and IgA. The BMI of the study group increased from 13.9+/-0.6 to 15.3+/-0.7 kg/m(2) (P = 0.02) with no significant change in the control group (14.1+/-0.7 and 14.5+/-0.5, respectively, P = 0.81). The percentage of resuming work was higher in the study group than in the control group. CONCLUSION: Perioperative parenteral nutrition possibly ameliorates the humoral immunity, reverses malnutrition, and facilitates rehabilitation.

[Detection of eotaxin and its clinical diagnosis value in patients with bronchial asthma].

OBJECTIVE: To explore the role of eotaxin in the pathogenesis of bronchial asthma and the clinical value in the diagnosis of asthma. METHODS: Serum eotaxin were measured by ELISA in 38 patients with asthma, 28 patients with non-asthma allergy, and 30 healthy controls. RESULTS: The levels of serum eotaxin in the asthma group were higher than those in the non-asthma allergic and control group (P<0.01). Furthermore, eotaxin levels in patients with acute asthma were significantly higher than those in patients with stable asthma (P<0.001). It was also found that the eotaxin levels of the acute asthma group were positively correlated to the amounts of eosinophils in peripheral blood (r=0.4196, P<0.001), and inversely correlated to the forced expiratory volume in one second (FEV1) (r=-0.3746, P<0.001). CONCLUSION: It suggests that eotaxin may play a crucial pathogenic role in the asthmatic process possibly by activating the allergic inflammatory cells and controlling the recruitment of eosinophils from blood to bronchial epithelium of the airway. The concentration of eotaxin is significantly associated with the attack of acute asthma and its severity. Eotaxin may be a potential therapeutic target in patients with asthma.

[Preparation of monoclonal antibodies against SARS coronavirus and staining usage in lung autopsy specimens].

OBJECTIVE: To prepare monoclonal antibodies against SARS coronavirus (SARS-CoV) on the purpose to explore the diagnosis methods of SARS. METHODS: Female BALB/C mice were immunized with disinfected SARS-CoV (PUMC01) and the spleen cells were fused with myeloma NS-1 cells. The hybridoma cell strains were screened by enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA) and Western blotting. Autopsy lung tissue sections from SARS patients were stained with ascites of monoclonal antibody (M2 strain) by immunohistochemical technique. RESULTS: Six strains of hybridomas that produced the monoclonal antibodies against SARS-CoV were obtained. The hybridomas were tested to have specific reactions with SARS-CoV and no cross-reactivates with other common respiratory disease causing pathogens. Of the 6 strains, 1 was identified as the immunoglobulin G3 (IgG3) isotype, 5 were IgG1. Western blotting showed that one strain (M2) reacted with 68000- Dalton (68KD) protein and four strains with 27000-dalton (27KD) protein. Band of M4 stain was not got by western blotting. Brown particles were seen in macrophages and pneumocytes in autopsy lung tissues from SARS patients. CONCLUSION: Monoclonal antibodies we made were specific to the SARS-CoV, and they had been used to detect SARS-CoV in autopsy lung tissues specimens with positive results.

[Analysis on the SARS-CoV genome of PUMC01 isolate].

OBJECTIVE: To perform variation and phylogenetics analysis on the SARS-CoV genome sequence (PUMC01) isolated in the Peking Union Medical College Hospital. METHODS: The cDNA library of SARS-CoV (PUMC01 isolate) was constructed by means of random-priming strategy. Random selected plasmid was sequenced and the genome sequence of SARS-CoV-PUMC01 was assembled by conventional methods (The Genebank Accession No. of SARS-CoV-PUMC01 is AY350750). The variation and phylogenetics analysis were performed by comparing the PUMC01 sequence with other SARS-CoV isolates. RESULTS: Ten variation sites were found by comparing PUMC01 isolate with Tor2 and Urbani isolates. In phylogenetic analysis of 18 SARS-CoV isolates, two classes were observed and there is different differential time between these two classes and the different isolates in each class. CONCLUSIONS: The evidence of phylogenetic analysis of different SARS-CoV isolates from different region is instructive for understanding the clinical relations between the different isolates and the transmission chain of SARS-CoV.

[cDNAs cloning of SARS-CoV PUMC2 viral genome].

OBJECTIVE: To get the cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain. METHODS: Using the SARS-CoV PUMC2 strain genomic RNA as the template, the cDNA fragments were amplified by RT-PCR, the PCR products were further purified and ligated into the pGEM-T vector, and all the clones obtained were sequenced. RESULTS: The cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain were obtained. CONCLUSIONS: These cDNAs can be provided for the function study of SARS-CoV proteins and the construction of full-length infectious cDNA clone of SARS-CoV.

[Cloning, expression and purification of SARS coronavirus PUMC2 strain nucleocapsid protein].

OBJECTIVE: To clone, express and purify nucleocapsid protein from SARS coronavirus PUMC2 strain. METHODS: According to the published SARS coronavirus genome sequences, the full length cDNA of N protein from SARS coronavirus PUMC2 strain was cloned by RT-PCR and the cDNA was cloned into the pET32a expression vector. The recombinant N protein was expressed in E. coli BL21 (DE3), and purified by Ni(2+)-NTA. RESULTS: Prokaryoticly expressed and purified N protein of SARS coronavirus PUMC2 strain was obtained. CONCLUSIONS: The SARS coronavirus recombinant N protein obtained by genetic engineering methods can be used for further functional study of SARS coronavirus N protein.

[Isolation and identification of SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed SARS patients].

OBJECTIVE: To isolate and identify SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed severe acute respiratory syndrome (SARS) patients. METHODS: Nasal and throat swab specimens were inoculated onto well of 24-well plate containing confluent monolayers of Vero and MRC-5 cells. Isolates were identified with serology, electron microscopy and genome sequence. RESULTS: One hundred and fifty-eight nasal and throat swabs specimens from 79 SARS patients in Peking Union Medical College Hospital between April and May, 2003 were cultured for SARS-coronavirus. Cytopathic effect (CPE) was found in three nasal swab specimens inoculated in Vero cells. Acute and convalescent phase serum specimens collected from SARS patients were found with seroconversions and/or a fourfold or greater rises in indirect fluorescence antibodies (IgG and IgM) titers when the 3 isolates (infected Vero cells) were used as antigen. Coronavirus was observed in the culture supernatant by negative-stain electron microscopy. Genome sequence confirmed the isolates were SARS-coronavirus. CONCLUSIONS: The 3 isolates from nasal and throat swabs samples collected from 79 clinically diagnosed SARS patients were SARS coronavirus.

[Reliability of detecting SARS-CoV antibody for diagnosis of SARS].

OBJECTIVE: To discuss the reliability of SARS-CoV antibody detection for SARS diagnosis. METHODS: Using SARS-CoV ELISA kit to detect relevant antibody in fresh serum of healthy, fever, probable, and suspect cases. RESULTS: The positive rate is 0%, 40%, and 95% respectively in healthy, probable, and suspect cases. CONCLUSIONS: It is reliable to detect SARS-CoV antibody in late suspect patients, but there will be high false-positive result in ordinary fever cases.

Expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome.

In a cohort of 38 patients with severe acute respiratory syndrome (SARS), we observed leukopenia in 47% of patients, lymphopenia in 84%, and T lymphopenia in 95%. CD4(+) T lymphocyte levels were reduced in 100% of patients, CD8(+) T lymphocyte levels were reduced in 87%, B lymphocyte levels were reduced in 76%, and natural killer cell levels were reduced in 55%. Our data suggested that these patients' immune systems were impaired during the course of SARS. The absolute counts of lymphocyte subsets demonstrated a clinical significance for patients with SARS.

[Clinical features and therapy of 106 cases of severe acute respiratory syndrome].

OBJECTIVE: To investigate the clinical characteristics of severe acute respiratory syndrome (SARS) and find out its effective treatment. METHODS: A total of 106 cases of SARS were analyzed prospectively. RESULTS: In this group, 56 were male and 50 female, aged from 15 to 81 years [average (36 +/- 10) years]. Common symptoms included fever (98.1%), chills (75.5%), cough (71.7%), headache and breathless (both 43.4%), diarrhea (24.5%) and rare rales in the lungs (11.2%). Laboratory test showed leukopenia (34.0%), lymphopenia (81.1%) and an extraordinary decrease of CD(4)(+) T cells (98.1%). Other rare abnormalities included liver injury (elevated alanine aminotransferase in 7.6%) and thrombocytopenia (3.8%). Almost all patients suffered from hypoxemia (PaO(2) less than 90 mm Hg in 90.2%, less than 70 mm Hg in 28.6%). Chest radiographs showed that unilateral focal patchy involvement in 34.0% of the patients, and unilateral multifocal or bilateral involvement were 11.3% and 46.2% respectively. Treatment regimens included small doses of steroids (methylprednisolone 40-80 mg, q12 h recommended) accompanied with broad-spectrum antibiotics such as the second generation of cephalosporins and macrolides and some other antiviral drugs. Meanwhile, emphasis was placed on oxygen support and coping with their underlying diseases. CONCLUSIONS: SARS has various presentations of clinical features and laboratory tests. Detection of CD(4)(+) T cell count is beneficial to diagnose SARS in early stage. Effective treatment includes various regimens, oxygen support and small doses of steroids.

[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.