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1、Mig as a Mediator for Brain Damage Caused by SARS CID 2005:41 (15 October) 1089M A J O R A R T I C L EDetection of Severe Acute Respiratory SyndromeCoronavirus in the Brain: Potential Role of theChemokine Mig in PathogenesisJun Xu,1Shuqing Zhong,1Jinghua Liu,2Li Li,1Yong Li,1Xinwei Wu,3Zhijie Li,2Pe
2、ng Deng,2Jingqiang Zhang,4Nanshan Zhong,1Yanqing Ding,2and Yong Jiang21Guangzhou Institute of Respiratory Diseases,2Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University,3Guangzhou Center for Diseases Control and Prevention, and4Electronic Microscope Center, Sun
3、Yatsen University, Guangzhou,Peoples Republic of ChinaBackground.Previous studies have shown that common human coronavirus might be neurotropic, althoughit was first isolated as a pathogen of the respiratory tract. We noticed that a few patients with severeacuterespiratorysyndrome (SARS) experienced
4、 central nervous symptoms during the course of illness. In the present study, weisolated a SARS coronavirus strain from a brain tissue specimen obtained from a patient with SARS with significantcentral nervous symptoms.Methods.Using transmission electronic microscopy and nested reverse transcription
5、polymerasechainreaction,the causative pathogen was identified in cultures of a brain tissue specimen obtained from the patient with SARS.Histopathologic examination of the brain tissue was performed using the methods of immunohistochemistryanalysis and double immunofluorescence staining. Fifteen cyt
6、okines and chemokines were detected in the bloodof the patient with SARS by means of a bead-based multiassay system.Results.A fragment specific for SARS human coronavirus was amplified from cultures of the brainsuspension,and transmission electronic microscopy revealed the presence of an enveloped v
7、irus morphologically compatiblewith a coronavirus isolated in the cultures. Pathologic examination of the brain tissue revealed necrosis of neuroncells and broad hyperplasia of gliocytes. Immunostaining demonstrated that monokine induced by interferon-g(Mig) was expressed in gliocytes with the infil
8、tration of CD68+monocytes/macrophages and CD3+T lymphocytesin the brain mesenchyme. Cytokine/chemokine assay revealed that levels of interferon-ginducible protein 10 andMig in the blood were highly elevated, although the levels of other cytokines and chemokines were close to normal.Conclusions.This
9、study provides direct evidence that SARS human coronavirus is capable of infecting thecentral nervous system, and that Mig might be involved in the brain immunopathology of SARS.The causative pathogen of severe acute respiratory syn-drome (SARS) has been identified as a new member ofthe coronavirus
10、family that exhibits a broad range ofhosts,infectingmanymammalianandavianspeciesandcausing upper respiratory, gastrointestinal, hepatic,andCNS diseases 1, 2. Two known human coronaviruses,229E and OC43, which cause up to one-third of allcases of common cold, were also found to infect theReceived 21
11、February 2005; accepted 14 June 2005; electronically published12 September 2005.Reprints or correspondence: Dr. Yong Jiang, Key Laboratory of Functional,Proteomics of Guangdong Province, Dept. of Pathophysiology, Southern MedicalUniversity, Tonghe, Guangzhou 510515, Peoples Republic of China ().Clin
12、ical Infectious Diseases2005;41:108996? 2005 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2005/4108-0003$15.00CNS 3. Our recent study of the SARS epidemic foundthat the spike protein, a surface antigen determiningthe tropism of coronavirus, had the strongest responset
13、o positive selection pressure 4. We noticed that a fewpatients with SARS in hospitals in Guangzhou City,China, exhibited central nervous symptoms during thecourse of their illnesses. Lauandcolleagues5reportedthat a CSF sample obtained from a 32-year-old womanwith SARS tested positive for SARS corona
14、virus(SARS-CoV) by RT-PCR, indicating that SARS-CoV mightcause an infection in the CNS of patients with SARS.In the present study, we isolated a SARS-CoV strainfrom a brain tissue specimen obtained during autopsyfrom a patient with SARS who became severely sickand showed significant central nervous
15、symptoms dur-ing the course of his illness. Furthermore, we investi-gated the immunopathological mechanism of brain at Florida International University on June 14, 2015http:/cid.oxfordjournals.org/Downloaded from 1090 CID 2005:41 (15 October) Xu et al.damage in the patient with SARS. In this study,
16、we detected ahigh level of monokine induced by IFN-g (Mig), a member ofCXC family 6, 7, in the patients brain and found that glio-cytes were a major source for Mig production in the brain. Allresults suggested that Mig was involved in the immunopath-ology of the brain on invasion of SARS-CoV.CASE RE
17、PORTA 39-year-old doctor whowasinchargeoftreatmentofpatientswith SARS in the respiratory intensive care unit of the ChestHospital (Guangzhou, China) developed a sudden onset offever, chills, malaise, headache, dizziness, and myalgia and washospitalized on 2 April 2003. Physical examination showed at
18、emperature of 38.5?C, clear lungs, and no obvious focus ofinfection. His fever resolved with treatment with ribavirin andmethylprednisolone (dosage, 120 mg per day), and his con-dition improved during the first week of treatment. A chestradiograph obtained on day 11 after the onset of symptomsreveal
19、ed left lower lobe infiltrates. A complete blood countdemonstrated an elevated WBC count ofcells/L, an916.4?10increased level of neutrophils (cells/L), and lym-915.5?10phopenia (lymphocyte count,cells/L). This suggests90.4?10that the condition of the patient with SARS was complicatedby a secondary b
20、acterial infection. Treatment with methyl-prednisolone was switched to 160 mg per 12 h intravenously,coincident with the initiation of antibiotic therapy. After ini-tiation of the treatment mentioned above, his condition grad-ually improved during the fourth week after onset, at whichtime a series o
21、f chest radiographs showed obvious resolutionof the left lower lobe consolidation. His total WBC count re-turned to a normal level, but lymphopenia still persisted.Treat-ment with methylprednisolone was then reduced to 40 mg per12 h. For the patients complaint of obscuredmonocularvision,eye-ground e
22、xamination was performed and revealed an exu-dation around the visual yellow zone on day 26 after onset ofillness (27 April). Two days later, the patient experienced pro-gressive central nervous symptoms, including dysphoria, vom-iting, and deliria, and was found to have progression of leftlower lob
23、e consolidation, for which he was treated with non-invasive ventilation and methylprednisolone at a dosage of 80mg per 12 h, coincident with initiation of treatment with cef-tazidime (Fortum; Glaxo Wellcome) and immunoglobulin.De-spite the management described above, his condition deteri-orated, and
24、 a series of chest radiographs obtained during thefifth week after onset showed progressive bibasilar infiltrationwith severe leucopenia (WBC count,/L), lympho-90.86?10penia (lymphocyte count,cells/L), and depressed90.11?10marrow.After receiving intravenous sedative, the patient developedcoma on day
25、 33 of illness, after which he was transferred intothe respiratory intensive care unit of the Guangzhou Instituteof Respiratory Diseases (Guangzhou, China). A CT scan of thebrain revealed broad encephalic pathological changes of prob-ably ischemia and necrosis and brain edema (figure 1). By day2 aft
26、er admission to the Guangzhou Institute of RespiratoryDiseases, the patient developed breathness with slowing heart-throb; thus, he underwent intubation and received mechanicalventilation. His condition rapidly deteriorated; he experiencedmultiorgan dysfunction syndrome, and brain herniation oc-curr
27、ed. He died on day 3 after his admission to the GuangzhouInstitute of Respiratory Diseases (35 days after the onset ofillness). Serum specimens obtained from the patient at earlyand later phases of the illness were forwarded to the Centersfor DiseaseControlandPrevention(Guangzhou,China),wherethe dia
28、gnosis of SARS was confirmed by ELISA and indirectfluorescence assay 8.METHODSAutopsy materials.Full autopsy was performed on the pa-tients with SARS. A detailed pathologic examination was per-formed on the brain tissue obtained during autopsy.Aparaffin-embedded block of the brain tissue was section
29、ed at a thicknessof 5 mm and dewaxed according to standard procedures.Immunohistochemistry and immunofluorescence staining.Histopathologic evaluation was performed on the specimen ofbrain tissue obtained during autopsy. Immunostainingwasper-formed with monoclonal antibodies to N protein of SARS-CoV(
30、supplied as a gift from Dr. Xiaoyan Che, Zhujiang Hospital,Southern Medical University, China), INF-ginducible protein10 (IP-10; Abcam), CD3 and CD68 (DAKO), and a polyclonalantibody to Mig (Abcam). To identify the cell type with ex-pression of Mig, double immunofluoresence staining was per-formed w
31、ith a primary polyclonal antibody to Mig plus amonoclonal antibody to glial fibrillary acidic protein (Chem-icon International) and secondary goat antirabbit antibodyconjugated with tetramethylrhodamineisothiocyanateandgoatantimouse antibody conjugated with fluoresceinisothiocyanate(Sigma-Aldrich).I
32、solation and identification of SARS-CoV.We inoculatedthe suspension of the brain tissue onto 2 cell lines: Vero-e6(C1008; ATCC) and human embryo lung fibroblast. The initialcytopathic effect (CPE) was observed between days 3 and 5.The CPE was a refractive appearance of cell rounding followedby cell
33、detachment and quickly spread to the entire cell mono-layer within 2448 h after the initiation of CPE. Specimenswere prepared for electronic microscopy by fixing a washed cellpellet with 2.5% glutaraldehyde and embedding it with epoxyresin.RT-PCR.Nucleic acids were purified from the superna-tants of
34、 cell cultures with CPE using a nucleic acid extractionkit (Amplimedical SpA-Bioline Division). The cDNA synthesisfrom the extracted RNA was performed using a reverse-tran- at Florida International University on June 14, 2015http:/cid.oxfordjournals.org/Downloaded from Mig as a Mediator for Brain Da
35、mage Caused by SARS CID 2005:41 (15 October) 1091Figure 1.A CT scan of different brain sections shows a diffuse brain edema with multiple high-density lesions (AF). Arrows, high-density lesions.Table 1.Results of general laboratory examination of the patient with severe acute respiratory syndrome(SA
36、RS), by number of days after onset of illness.Laboratory measurementDay after onset of SARS111212931333435WBC count, ?109cells/L4.7216.405.8721.017.013.411.510.86Lymphocyte count, ?109cells/L0.720.400.480.320.320.310.240.11Neutrophil count, ?109cells/L3.4615.504.9520.226.242.801.070.66Platelet count
37、, ?109cells/L212160453737252628LDH level, U/L1513262084584644655211233AST level, U/L2635372420121314ALT level, U/L32103866043302928CRP level mg/L2.9124.124.9298.6278.0306.7418.0417.8NOTE.ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP , C-reactive protein; LDH, lactate dehydrogen
38、ase.scription kit (Amplimedical SpA-Bioline Division). The pro-cedure is based on the reverse-transcription reaction utilizingtherandomprimertechnique.AnestedRT-PCRwasperformedin accordance with the protocol developed by Dr. ChristianDrosten with outerprimers5?-ATGAATTACCAAGTCAATGG-TTAC-3?and 5?-CAT
39、AACCAGTCGGTACAGCTA-3?and in-nerprimers 5?-GAAGCTATTCGTCACGTTCG-3?and 5?-CTG-TAGAAAATCCTAGCTGG AG-3?8.Cytokine/chemokine assay.Quantification of multiple cy-tokines/chemokines was performed using a LiquiChip work-station (Qiagen GMBH), which is a bead-based system forimmunoassays that allows simultan
40、eous assays of multiple an-alytes from a single sample 9. A blood sample obtained fromthe patients with SARS on the day before death was used forthe cytokine/chemokine assay. The 15 cytokines/chemokinesassayed in this study included the following: IL-1b, IL-2, IL-4,IL-6, IL-8, IL-10, IL-12, TNF-a, I
41、FN-g, granulocyte macro-phagecolony-stimulating factor, Mig, IP-10, monocyte che-moattractant protein 1, macrophage inflammatory protein 1a,and regulated on activation, normal T cell expressed and se- at Florida International University on June 14, 2015http:/cid.oxfordjournals.org/Downloaded from 10
42、92 CID 2005:41 (15 October) Xu et al.Figure 2.Histochemistry stains showing neuron denaturation and necrosis with striated encephalomalacia (A; original magnification, ?100)(B; original magnification, ?400), vasocongestion with widening peripheral space the around vessel (C; original magnification,
43、?200), and focalhemorrhage (D; original magnification, ?100) and gliosome formation in the brain tissue (E; original magnification, ?200). Arrows, pathologicalalterations described above.creted (RANTES). Serum samples were obtained from 10healthy control subjects aged 2251 years. Samples were ana-ly
44、zed on the LiquiChip system (Qiagen GMBH), according tothe manufacturers instruction. Approval of the local ethicscommittees and informed consent were obtained.RESULTSGeneral laboratory examination.General laboratory ex-amination revealed a peak C-reactive protein level of 418.0mg/L and an increase
45、in the lactate dehydrogenase level to1233.0 U/L between days 1 and 35 after the onset of illness.Detailed information is shown in table 1.Histopathologic examination.Histopathologic examina-tion revealed invasive Aspergillus pneumonia with multiple ab-scess, constitutional diffuse mycohemia, and fun
46、gal multi-abscess in multiple organs. Pathologic examination of the braintissue specimen under microscope revealed neuron denatura-tion and necrosis, broad gliocytes hyperplasia with gliosomeformation, and encephalic edema (figure 2).Identification of SARS-CoV in the brain.The result ofthe nested RT
47、-PCR showed that a predicted cDNA fragment(BNI-1) of 108 bp specific for SARS-CoV 8 was amplifiedfrom Vero-E6 cell culture inoculated with the brainsuspension,but not from the culture that included medium only (figure3A). Examination by transmission electronic microscopy re-vealed the presence of en
48、veloped virus particles with a diameterof 8090 nm and a surface morphology compatible with acoronavirus (figure 3B) in the culture showing CPE 8.Immunohistochemistryanddouble immunofluoresencestaining.Immunohistochemistry staining of gliocytes andneurocytes obtained from the brain of the patient wit
49、h SARSrevealed them to be positive for N proteins (figure 4A), butthose obtained from a patient who died in a traffic accidentwere not (figure 4B). We found that Mig, but not IP-10, ex-pressed with infiltration of CD68+monocytes/macrophagesandCD3+T lymphocytes in the brain mesenchyme of the patient.
50、Double immunofluoresence staining revealed that Mig wasmainly expressed in gliocytes (figure 5). at Florida International University on June 14, 2015http:/cid.oxfordjournals.org/Downloaded from Mig as a Mediator for Brain Damage Caused by SARS CID 2005:41 (15 October) 1093Figure 3.Identification of
51、severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in the brain tissue of the patient with SARS.A, Results of nested RT-PCR for BNI-1 fragment (108 base pairs) of SARS-CoV in the supernatants obtained from cultured Vero-E6 cells with cytopathiceffect. Lane 1, DNA standard; lane 2, Vero-
52、E6 cell culture inoculated with the lung suspension; lane 3, Vero-E6 cell culture used as negative control;lane 4, Vero-E6 cell culture inoculated with the brain suspension. B, Transmission electronic microscopy reveals the presence of enveloped virusparticles with morphology compatible with coronav
53、irus. Extracellular particles were found clustering and adhering to the surface of the plasmamembrane.Figure 4.Immunohistochemistry stains for N protein of severe acuterespiratory syndrome (SARS) coronavirus (SARS-CoV) in a specimen ofbrain tissue obtained from the patient with SARS during autopsy.
54、Amonoclonal antibody against N protein of SARS-CoV and the secondaryantibody of horseradish peroxidase(HRP)conjugatedgoatantimousewereused for the immunohistochemical staining of brain tissue specimensobtained from the patient with SARS (A; original magnification, ?200)and a patient who died in a tr
55、affic accident (B; original magnification,?200). Staining was performed with diaminobenzidine (brown). Arrows,positive staining cells.Cytokine/chemokine profile in the blood.As shown infigure 6, the levels of IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-a, IFN-g, granulocyte macrophagecolony-sti
56、mulat-ing factor, RANTES,macrophageinflammatoryprotein1a,andmonocyte chemoattractant protein 1 were close to those of thecontrol subjects, but the levels of IP-10 and Mig in the bloodof the patient with SARS were highly elevated.DISCUSSIONOn review of the patients vitalrecord,wefoundthathisgeneralco
57、ndition was nearly normal, and a chest radiograph showedresolving left lower pulmonary infiltration when he developedinitial central nervous symptoms with an exudation aroundthevisual yellow zone, indicating that the pathologic change in thebrain was independent of pulmonary superinfection in the pa
58、-tient. In addition to findings of CT scanning indicating braindamage, pathologic examination revealed gliocyteshyperplasia,neuron denaturation, and necrosis combined with a striatedencephalomalacia, suggesting that a chronic progressive viralcerebritis was present in the patient during the course o
59、f illness.Neuroinvasion by SARS-CoV was also directly evidenced bythe viral morphology observed under electronic microscope,genetic identification, and the viral antigen (N protein) foundin the brain.Previous studies found that coronavirus might be neuro-tropic, neuroinvasive, and neurovirulent, alt
60、hough it was firstisolated as a pathogen of the respiratory tract 1, 3. At least3 routes, including olfactory nerve, a hematogenous route, andlymphatic systems could be used by coronavirus to gain accessto the CNS 1012. The cell lines of astrocytoma, neuroblas-toma, neuroglioma, and oligodendrocyte
61、were all susceptibleto the infection of human coronaviruses 1316. Mouse hep-atitis virus, a murine coronavirus, has been found to inducedemyelinating disease of the CNS 17, 18. Moreover, Glassand colleagues 19 recently reported a model of SARS-CoV at Florida International University on June 14, 2015
62、http:/cid.oxfordjournals.org/Downloaded from 1094 CID 2005:41 (15 October) Xu et al.Figure 5.Immunohistochemistry and double immunofluoresence staining of specimens of brain tissue sections obtained from the patient withsevere acute respiratory syndrome during autopsy. Antibodies against IFN-ginduci
63、ble protein 10 (IP-10) (A; original magnification, ?200), monokineinduced by IFN-g (Mig) (B; original magnification, ?200), CD68 (C; original magnification, ?200), and CD3 (D; original magnification, ?200) wereused in the immunohistochemical staining. Antibodies against Mig (E; original magnificatio
64、n, ?400) and glial fibrillary acidic protein (F; originalmagnification, ?400) were used in double immunofluoresence staining. A, Brain tissues without evidence of IP-10 from staining. B, Mig-positivestaining cells detected in the brain tissue. C, Infiltration of CD68+monocyte/macrophage cells. D, A
65、few CD3+T lymphocytes. EG, A doubleimmunofluoresence stain showing that glial fibrillary acidic protein positive cells (F) were expressing Mig (E) and displaying yellow immunofluoresence(G; original magnification, ?400) when the 2 images were merged. Arrows, positive staining cells. AD were stained
66、by diaminobenzidine. EG werestained by both tetramethylrhodamine isothiocyanate (red) and FITC (green), respectively.infection in C57BL/6 mice that demonstrated that SARS-CoVwas able to infect the brain. The finding that SARS-CoV in-fected the brain of the patient with SARS was consistent withfindin
67、gs of previous reports on neuroinvasion of the corona-virus family 1319.Genomic comparison of strains of SARS-CoV showed thatimmunological pressures influenced the evolution of SARS-CoV in the human population by a change of the spike protein4. However, DNA sequence analysis showed that there wasno
68、variation with significant substitution of amino acids on thespike protein of the SARS-CoV isolated from this patient withthe primary isolates of SARS-CoV 4. It is possible that SARS-CoV is characterized by behavioral abnormalities similar tothose in human coronaviruses OC43 and 229E, which have the
69、capacity to infect the CNS but do not necessarily yield severepathological symptoms 13, 16, because local immune sur-veillance limits viral replication on a large scale 20. On thebasis of the consideration that a progressive lymphopenia andleukopenia developed 21, 22 during the disease course of thi
70、spatient, we speculatethatlocalimmunesurveillanceinhisbrainwas removed by severe immunodepression 2123, leading tothe amplification of infectious viruses.With regard to the superinfection with invasive Aspergillusin the brain and other organs of the patient, we think thatsevere immunodepression resu
71、lting from the damage to theimmune system induced by SARS-CoV infection, combinedwith high-dosage treatment with a corticosteroid, provided ac-cess for conditional pathogens, causing a superinfection withinvasive Aspergillus in multiple organs 24. In this case, As-pergillus infection might have syne
72、rgized or deteriorated theharmful effects of the SARS-CoV infection in the brain 24,which brought about a more complicated clinical process.Our previous study showed that the IP-10 level, one of thenonELR CXC chemokines, was augmented markedly in theblood of patients in the early stage of SARS and r
73、emained ata high level during the course of illness 25. Consistent withthat finding, this study showed that IP-10 in the blood of thepatient with SARS was also highly increased. Furthermore, wefound that the level of Mig, another CXCR3 chemokine, washighly elevated in the blood of the patient with S
74、ARS. In con-trast to our finding, Wong et al. 26 reported that Mig couldnot be detected with significant increase in the bloodofpatientswith SARS. The difference between the findings of Wong andcolleagues and our results provides evidence for the hypothesis at Florida International University on Jun
75、e 14, 2015http:/cid.oxfordjournals.org/Downloaded from Mig as a Mediator for Brain Damage Caused by SARS CID 2005:41 (15 October) 1095Figure 6.The profile of cytokines and chemokines in the blood of thepatient with severe acute respiratory syndrome (SARS). Each cytokine/chemokine is expressed as a r
76、atio of that of the patient with SARS tothe average among control subjects. Of all the detected cytokines/che-mokines, only IFN-ginducible protein 10 (IP-10) and monokine inducedby IFN-g (Mig) showed highly increased production.that the high level of Mig in the blood of this patient withSARS was ass
77、ociated with the invasion of SARS-CoV in thebrain tissues. It has been reported that both Mig and IP-10were involved in host defense and immune damage by attract-ing activated T cells, NK cells, and monocytes that expressCXCR3 27. We could not detect a significant elevation in theIP-10 level, but we
78、 did detect a high expression of Mig in thebrain. This result is different from findings of our previousstudy, which revealed a high level of IP-10 in the lungs ofpatients with SARS. The results of histopathologicexaminationand immunostaining indicated that the invasion of SARS-CoVof brain tissues b
79、rought about the induction of Migingliocytes,which in turn attracted CD68+monocytes/macrophages andCD3+T lymphocytes to the sites of virus infection 27, 28.Infiltrated immune cells should be helpful to limit SARS-CoVinfection of the brain. However, because the virus could notbe eliminated effectivel
80、y while the patient was immunode-pressed, a high number of cytokines/chemokines released inresponse to SARS-CoV infection would contribute to the tissuedamage 29, 30. The brain damage caused by SARS-CoV in-fection might take the same immunopathological process thathappened in the lung tissues of pat
81、ients with SARS 25, al-though different chemoattractant factors were involved.In conclusion, to our knowledge, our findings are the firstdirect evidence that SARS-CoV has the ability to infect theCNSand thattheinfectioncausesimmunopathologicaldamagelead-ing to a more serious and complicated clinical
82、 syndrome. Mig,but not IP-10, may be involved in the brain immunopathologyby attracting immune effector cells to the site of virus infection.However, the specific mechanism for Mig as a key mediator inthe brain damage induced by SARS-CoV infection needs fur-ther investigation.AcknowledgmentsWe thank
83、 Ms. Jing Zhou, College of Foreign Language and InternationalCommunication, Southern Medical University (Guangzhou, China) forassistance in the preparation of the manuscript.Financial support.973 Program of China (2002CB513005 and2003CB514117) and the National Natural Science Foundation of China(399
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