人ACE2转基因小鼠对SARS-CoV的易感性及SARS发病机制的实验研究
摘要
严重急性呼吸综合征(severe acute respiratory syndrome,SARS)是本世纪初爆发的由一种新的冠状病毒引起的传染性疾病,主要的靶器官是肺脏,病死率达10%左右。SARS已经淡出人们的生活已有四年多了,目前的研究主要集中在SARS发病机制和抗病毒药物、疫苗的研发上,而这些研究的基础是可靠的SARS动物模型。已有文献报道非人灵长类(恒河猴、食蟹猴、绒猴、非洲绿猴)、小鼠(BABL/c、C57/B6、基因修饰小鼠)、仓鼠、狸子猫、雪貂等均能支持SARS-CoV的复制,其中有些动物显示了不同程度的病理变化,但在临床疾病和病理上仍缺乏与SARS病人的相似性。
据研究,SARS-CoV是通过其表面的S蛋白和靶细胞的表面受体血管紧张素转换酶2(ACE2)结合侵入宿主细胞的,这提示人ACE2(hACE2)转基因鼠有可能作为对SARS-CoV易感的动物模型,因此我们进行了hACE2转基因鼠对SARS-CoV易感性的实验研究并利用此动物模型探讨了SARS的发病机制。
本研究主要包括以下三个部分的实验内容:
1.hACE2转基因小鼠的制备和鉴定:选择小鼠ACE2(mACE2)启动子驱动的hACE2全长编码序列(Coding sequence,CDS)基因片断,通过显微注射的方法,制备携带hACE2基因的转基因小鼠。经PCR检测到4个阳性首建鼠;RT-PCR及Nested RT-PCR结果显示在其中一个Founder的子代鼠的肺、心、肾和小肠有hACE2基因表达;Realtime-PCR检测此Founder子代鼠转基因的拷贝数,结果显示为单拷贝转基因小鼠;Western blot结果显示hACE2蛋白在转基因小鼠的不同组织包括肺、心、肾和小肠均有表达;免疫组化显示,在转基因小鼠的肾脏近曲小管上皮细胞、肺脏小血管内皮细胞可见hACE2蛋白表达:无创性血压监测观察发现,hACE2转基因小鼠的心率、血压与野生鼠相比未见明显差异;病理组织学检查未发现转基因小鼠心、肺、肾等器官组织结构异常。
2.hACE2转基因小鼠对SARS—CoV易感性的实验:经鼻腔接种PUMC01株SARS-CoV后,野生型和转基因小鼠接种SARS—CoV后均未出现死亡现象。在接种后的第3天和第7天,RT-PCR和免疫荧光试验(IFA)结果显示,转基因小鼠肺组织上清的病毒载量和病毒滴度明显高于野生型小鼠(P＜0.05);在接种后的第7天,转基因小鼠的感染率明显高于野生型小鼠(P＜0.01);转基因小鼠出现典型的间质性肺炎,而且伴有肾脏、心脏、肝脏、脑组织和胃肠道等肺外组织的损伤。而部分野生型小鼠只在感染后第三天出现轻微的间质性肺炎。通过免疫组化染色,在转基因小鼠的肺脏上皮细胞和血管内皮细胞及大脑神经细胞可发现SARS-CoV抗原;接种病毒后在部分感染小鼠的血清中可检测到SARS-CoV特异性的抗体IgG和IgM,但转基因小鼠抗体阳性率低于野生型小鼠。上述资料表明,hACE2转基因小鼠对SARS-CoV的易感性明显高于野生型小鼠。
3.利用hACE2转基因小鼠探讨非特异性的免疫反应在SARS发病中的作用:应用ELISA的方法,观察到转基因小鼠接种病毒后,肺组织上清细胞因子IL-6、TNF-α和IFN-γ明显升高;Western Blot和免疫组化染色检测到转基因小鼠感染SARS-CoV后肺组织一氧化氮合酶(iNOS)表达明显增强,阳性颗粒主要位于肺泡巨噬细胞;比色法测定到转基因小鼠感染SARS-CoV后血浆中NO含量明显增多。
综上所述,hACE2在我们制备的转基因小鼠体内部分组织表达,尽管接种病毒后具有缺少病死率的局限性,但hACE2转基因小鼠的易感性明显高于野生型小鼠,病理变化与SARS病人相似。这种模型的建立对于研究SARS发病机制和抗病毒药物的评价具有重要的意义。
Severe acute respiratory syndrome(SARS),an infectious disease which pathogenic agent was a novel coronavirus,broke out in 2003 and rapidly spreaded to many countries in the world.The main target organ of SARS was lung and the fatality rate was approximately 10%.Animal models provided an important tool for studying SARS pathogenesis and evaluating the efficacy of potential drugs and vaccines.Established models for SARS infection included cynomologus macaques,ferrets,cats, mice,African green monkeys,and Golden Syrian hamsters.Some of them presented lung pathological changes of human SARS infection with different severity.However,most of them lacked comparability to SARS patients on clinical diseases and pathology.
The spike proteins of SARS-CoV bind to receptor on host cells,named angiotensin-converting enzyme-2(ACE2),and mediate viral entry.This indicates that a mouse transgenic for human ACE2(hACE2) could serve as an animal model for SARS infection.Thus,we produced hACE2 transgenic mice and observed its susceptibility to SARS-CoV.In addition, we detected changes of some cytokines in the inoculated mice.The main works were included below.
1.The production and identification of hACE2 transgenic mice:The mACE2 promoter driving hACE2 CDS fragments were introduced into the pronuclei of ICR fertilized ova using microinjection.PCR was used to screen for the transgene in the genomic DNA of potential transgenic mice. RNA analysis by RT-PCR and nested RT-PCR showed that hACE2 mRNA was transcribed in the lung,heart,kidney,and intestine of transgenic mice. Levels of transgene DNA in founder3 was a single copy in genome as determined by Realtime-PCR.Western blot indicated that hACE2 protein levels of the lung,heart,kidney,and intestine from transgenic mice were all higher than that from wild-type mice.By Immunohistochemistry,hACE2 protein was detected in epithelial cells of kidney and vascular endothelial cells of lung.The uninjured detection of blood pressure of transgenic mice showed that there was not difference between wild and transgenic mice.No organs abnormalities were found in transgenic mice under microscope.
2.The experiment of susceptibility of transgenic mice to SARS-CoV:After inoculated intranasally by PUMC01 strain of SARS-CoV,none of thirty-six challenged wildtype and transgenic mice was dead by one week post-infection.The results of RT-PCR and immunofluoresce assay(IFA) indicated that the replication of SARS-CoV in the lung homogenates from transgenic mice were higher than from wild-type mice by day 3 and 7 post-inoculation.The transgenic mice present severe and typical interstitial pneumonia accompanied by many extra-pulmonary organ damages.The wild mice just showed mild interstitial pneumonia in day 3 post-infection. By immunohistochemistry(IHC),the antigen of SARS-CoV was found in epithelial cells,vascular endothelial cells of lung and cerebral neurocytes of transgenic mice.The specific antibodies were found both in wild and transgenic mice.However,the positive number in transgenic mice is less than that in wild mice.The above results showed that the hACE2 transgenic mice were more susceptible to SARS-CoV than wild mice.
3.The study of mechanism of SARS on transgenic mice:By ELISA,we found that levels of TNF-α,IL-6 and IFN-γin the lung homogenates from transgenic mice were increased more markedly than wild mice after they were inoculated with SARS-CoV.The expression of inducible nitric oxide synthase(iNOS) in the lung of infected transgenic mice was higher than that of wild mice by Western blot and IHC.The positive signal was found in macrophages in the alveoli.The level of nitric oxide(NO) increased greatly in serum of transgenic mice.
To sum up,hACE2 was expressed in some tissues of transgenic mice. Although it had limitation of the absence of lethality,transgenic mice model were more susceptible than wild mice with resembling to SARS patients in pathology and should still aid the evaluation of anti-SARS-CoV drugs and vaccines,as well as the analysis of SARS pathogenesis by virus detection and systemic pathological studies.
据研究,SARS-CoV是通过其表面的S蛋白和靶细胞的表面受体血管紧张素转换酶2(ACE2)结合侵入宿主细胞的,这提示人ACE2(hACE2)转基因鼠有可能作为对SARS-CoV易感的动物模型,因此我们进行了hACE2转基因鼠对SARS-CoV易感性的实验研究并利用此动物模型探讨了SARS的发病机制。
本研究主要包括以下三个部分的实验内容:
1.hACE2转基因小鼠的制备和鉴定:选择小鼠ACE2(mACE2)启动子驱动的hACE2全长编码序列(Coding sequence,CDS)基因片断,通过显微注射的方法,制备携带hACE2基因的转基因小鼠。经PCR检测到4个阳性首建鼠;RT-PCR及Nested RT-PCR结果显示在其中一个Founder的子代鼠的肺、心、肾和小肠有hACE2基因表达;Realtime-PCR检测此Founder子代鼠转基因的拷贝数,结果显示为单拷贝转基因小鼠;Western blot结果显示hACE2蛋白在转基因小鼠的不同组织包括肺、心、肾和小肠均有表达;免疫组化显示,在转基因小鼠的肾脏近曲小管上皮细胞、肺脏小血管内皮细胞可见hACE2蛋白表达:无创性血压监测观察发现,hACE2转基因小鼠的心率、血压与野生鼠相比未见明显差异;病理组织学检查未发现转基因小鼠心、肺、肾等器官组织结构异常。
2.hACE2转基因小鼠对SARS—CoV易感性的实验:经鼻腔接种PUMC01株SARS-CoV后,野生型和转基因小鼠接种SARS—CoV后均未出现死亡现象。在接种后的第3天和第7天,RT-PCR和免疫荧光试验(IFA)结果显示,转基因小鼠肺组织上清的病毒载量和病毒滴度明显高于野生型小鼠(P＜0.05);在接种后的第7天,转基因小鼠的感染率明显高于野生型小鼠(P＜0.01);转基因小鼠出现典型的间质性肺炎,而且伴有肾脏、心脏、肝脏、脑组织和胃肠道等肺外组织的损伤。而部分野生型小鼠只在感染后第三天出现轻微的间质性肺炎。通过免疫组化染色,在转基因小鼠的肺脏上皮细胞和血管内皮细胞及大脑神经细胞可发现SARS-CoV抗原;接种病毒后在部分感染小鼠的血清中可检测到SARS-CoV特异性的抗体IgG和IgM,但转基因小鼠抗体阳性率低于野生型小鼠。上述资料表明,hACE2转基因小鼠对SARS-CoV的易感性明显高于野生型小鼠。
3.利用hACE2转基因小鼠探讨非特异性的免疫反应在SARS发病中的作用:应用ELISA的方法,观察到转基因小鼠接种病毒后,肺组织上清细胞因子IL-6、TNF-α和IFN-γ明显升高;Western Blot和免疫组化染色检测到转基因小鼠感染SARS-CoV后肺组织一氧化氮合酶(iNOS)表达明显增强,阳性颗粒主要位于肺泡巨噬细胞;比色法测定到转基因小鼠感染SARS-CoV后血浆中NO含量明显增多。
综上所述,hACE2在我们制备的转基因小鼠体内部分组织表达,尽管接种病毒后具有缺少病死率的局限性,但hACE2转基因小鼠的易感性明显高于野生型小鼠,病理变化与SARS病人相似。这种模型的建立对于研究SARS发病机制和抗病毒药物的评价具有重要的意义。
Severe acute respiratory syndrome(SARS),an infectious disease which pathogenic agent was a novel coronavirus,broke out in 2003 and rapidly spreaded to many countries in the world.The main target organ of SARS was lung and the fatality rate was approximately 10%.Animal models provided an important tool for studying SARS pathogenesis and evaluating the efficacy of potential drugs and vaccines.Established models for SARS infection included cynomologus macaques,ferrets,cats, mice,African green monkeys,and Golden Syrian hamsters.Some of them presented lung pathological changes of human SARS infection with different severity.However,most of them lacked comparability to SARS patients on clinical diseases and pathology.
The spike proteins of SARS-CoV bind to receptor on host cells,named angiotensin-converting enzyme-2(ACE2),and mediate viral entry.This indicates that a mouse transgenic for human ACE2(hACE2) could serve as an animal model for SARS infection.Thus,we produced hACE2 transgenic mice and observed its susceptibility to SARS-CoV.In addition, we detected changes of some cytokines in the inoculated mice.The main works were included below.
1.The production and identification of hACE2 transgenic mice:The mACE2 promoter driving hACE2 CDS fragments were introduced into the pronuclei of ICR fertilized ova using microinjection.PCR was used to screen for the transgene in the genomic DNA of potential transgenic mice. RNA analysis by RT-PCR and nested RT-PCR showed that hACE2 mRNA was transcribed in the lung,heart,kidney,and intestine of transgenic mice. Levels of transgene DNA in founder3 was a single copy in genome as determined by Realtime-PCR.Western blot indicated that hACE2 protein levels of the lung,heart,kidney,and intestine from transgenic mice were all higher than that from wild-type mice.By Immunohistochemistry,hACE2 protein was detected in epithelial cells of kidney and vascular endothelial cells of lung.The uninjured detection of blood pressure of transgenic mice showed that there was not difference between wild and transgenic mice.No organs abnormalities were found in transgenic mice under microscope.
2.The experiment of susceptibility of transgenic mice to SARS-CoV:After inoculated intranasally by PUMC01 strain of SARS-CoV,none of thirty-six challenged wildtype and transgenic mice was dead by one week post-infection.The results of RT-PCR and immunofluoresce assay(IFA) indicated that the replication of SARS-CoV in the lung homogenates from transgenic mice were higher than from wild-type mice by day 3 and 7 post-inoculation.The transgenic mice present severe and typical interstitial pneumonia accompanied by many extra-pulmonary organ damages.The wild mice just showed mild interstitial pneumonia in day 3 post-infection. By immunohistochemistry(IHC),the antigen of SARS-CoV was found in epithelial cells,vascular endothelial cells of lung and cerebral neurocytes of transgenic mice.The specific antibodies were found both in wild and transgenic mice.However,the positive number in transgenic mice is less than that in wild mice.The above results showed that the hACE2 transgenic mice were more susceptible to SARS-CoV than wild mice.
3.The study of mechanism of SARS on transgenic mice:By ELISA,we found that levels of TNF-α,IL-6 and IFN-γin the lung homogenates from transgenic mice were increased more markedly than wild mice after they were inoculated with SARS-CoV.The expression of inducible nitric oxide synthase(iNOS) in the lung of infected transgenic mice was higher than that of wild mice by Western blot and IHC.The positive signal was found in macrophages in the alveoli.The level of nitric oxide(NO) increased greatly in serum of transgenic mice.
To sum up,hACE2 was expressed in some tissues of transgenic mice. Although it had limitation of the absence of lethality,transgenic mice model were more susceptible than wild mice with resembling to SARS patients in pathology and should still aid the evaluation of anti-SARS-CoV drugs and vaccines,as well as the analysis of SARS pathogenesis by virus detection and systemic pathological studies.
引文
1 World Health Organization (WHO). Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003.http://www.who.int/csr/sars/country/table2004_04_21/en/, 2003 (accessed Feb 3, 2005).
2 Kuiken, T. Fouchier RA, Schutten M, et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 2003; 362:263-270.
3 Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348:1967-1976.
4 Tsang KW, Ho PL, Ooi GC, et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1977-1985.
5 He JF, Xu RH, Yu DW, et al. Severe acute respiratory syndrome in Guangdong Province of China: epidemiology and control measures.Zhonghua Yu Fang Yi Xue Za Zhi. 2003; 37: 227-232.
6 Bukreyev A, Lamirande EW, Buchholz UJ, et al. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 2004; 363:2122-2127.
7 Lawler JV, Endy TP, Hensley LE, et al. Cynomolgus macaque as an animal model for severe acute respiratory syndrome. PLoS Med 2006; 3:el49.
8 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425:915.
9 Qin C, Wang J, Wei Q, et al. An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus. J Pathol 2005;206:251-259.
10 Roberts A, Paddock C, Vogel L, et al. Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans. J Virol 2005; 79:5833-5838.
11 Roberts A, Vogel L, Guarner J, et al. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters. J Virol 2005; 79:503-511
12 Rowe T, Gao G, Hogan RJ, et al. Macaque model for severe acute respiratory syndrome. J Virol 2004; 78:11401 -11404.
13 Babcock GJ, Esshaki DJ, Jr Thomas WD, et al Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol 2004; 78:4552-4560.
14 Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003; 426:450-454.
15 Wang P, Chen J, Zheng A, et al. Expression cloning of functional receptor used by SARS coronavirus. Biochem Biophys Res Commun 2004;315:439-444.
16 McCray PB Jr, Pewe L, Wohlford-Lenane C, et al. LETHAL INFECTION IN K18-hACE2 MICE INFECTED WITH SARS-CoV. J Virol 2007;81:813-821.
17 Tseng CT, Huang C, Newman P, et al. SARS Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 (hACE2) Virus Receptor.J Virol 2007; 81:1162-1173.
18 Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG,Birnberg NC, Evans RM. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature.1982 Dec 16; 300 (5893):611-615.
19 Baeumler S, Wulff D, Tagliani L, Song P. A real-time quantitative PCR detection method specific to widestrike transgenic cotton (event 281-24-236/3006-210-23). J Agric Food Chem. 2006 Sep 6;54(18):6527-6534.
20 Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB,Mourrain P, Palauqui JC, Vernhettes S. Transgene-induced gene silencing in plants. Plant J. 1998 Dec;16 (6):651-659.
21 Hamming, Timens W, Bulthuis MLC, et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004; 203:631-637.
22 Crackower MA, Sarao R, Oudit GY, et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 2002; 417:822-828.
23 Donoghue M, Hsieh F, Baronas E, et al. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res 2000; 87:E1-E9.
24 Mary Donoghue, Hiroko Wakimoto, Colin T Maguire, et al. Heart block,ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins. J Mol Cell Cardiol. 2003 Sep ;35 (9): 1043-1053
25 Subbarao K, McAuliffe J, Vogel L, et al. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J Virol 2004;78:3572-3577.
26 Leung TF, Wong GW, Hon KL, Fok TF. Severe acute respiratory syndrome (SARS) in children: epidemiology, presentation and management. Paediatr Respir Rev. 2003 Dec; 4 (4):334-339.
27 Rainer TH, Chan PK, Ip M, Lee N, Hui DS, Smit D, Wu A, Ahuja AT, Tarn JS,Sung JJ, Cameron P. The spectrum of severe acute respiratory syndrome-associated coronavirus infection. Ann Intern Med. 2004 Apr 20;140 (8):614-619.
28 Osterhaus AD, Fouchier RA, Kuiken T. The aetiology of SARS: Koch's postulates fulfilled. Philos Trans R Soc Lond B Biol Sci 2004; 359:1081-82.
29 Fouchier RA, Kuiken T, Schutten M, et al. Aetiology: Koch's postulates fulfilled for SARS virus. Nature 2003; 423:240.
30 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425:915.
31 Wentworth DE, Gillim-Ross L, Espina N, Bernard KA. Mice susceptible to SARS coronavirus. Emerg Infect Dis 2004; 10:1293-1296.
32 Glass WG, Subbarao K, Murphy B, Murphy PM. Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 2004;173:4030-4039.
33 Hwang DM, Chamberlain DW, Poutanen SM, et al. Pulmonary pathology of severe acute respiratory syndrome in Toronto. Mod Pathol 2005; 18:1-10.
34 Lang ZW, Zhang LJ, Zhang SJ, et al. A clinicopathological study of three cases of severe acute respiratory syndrome (SARS). Pathology 2003;35:526-531.
35 Ding Y, Wang H, Shen H, et al. The clinical pathology of severe acute respiratory syndrome(SARS):a report from China.d Pathol 2003;200:282-289.
36 Jeffers,S.A.,Tusell,S.M.,Gillim-Ross,L.,Hemmila,E.M.,Achenbach,J.E.,Babcock,G.J.,Thomas,W.D.Jr.,Thackray,L.B.,Young,M.D.,Mason,R.J.,Ambrosino,D.M.,Wentworth,D.E.,DeMartini,J.C.,Holmes,K.V.(2004).CD209L(L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus.Proc.Natl.Acad.Sci.U.S.A.101:15748-15753.
37 石玉玲 李林海 徐德兴 等.广东地区严重急性呼吸综合征(SARS)患者血清IFA检测[J].细胞与分子免疫学杂志,2003,19(5):封2.
38 石玉玲 李林海 石凌波 张亚松 徐德兴万根平顾晓琼 张小玲.广州地区住院患儿血清抗SARS病毒抗体的检测[J].细胞与分子免疫学杂志,2003年19卷6期
39 崔小岱 宋国维 马连华 许峥 王天有.儿童SARS病毒抗体回顾检测及其意义[J].中华医学研究杂志,2004年第4卷第3期
40 Nicholls,J.M.,Lee,K.C.,Lee,K.C.(2003).Lung pathology of fatal severe acute respiratory syndrome.LANCET 361:P 1773-1778.
41 Wong CK,Lam CW,Wu AK,Ip WK,Lee NL,Chan IH,Lit LC,Hui DS,Chan MH,Chung SS,Sung JJ.Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome.Clin Exp Immunol.2004Apr;136(1):95-103.
42 Xie J,Han Y,Li TS,Qiu ZF,Ma X J,Fan HW,Lu W,Liu ZY,Wang Z,Wang HL,Deng GH.Dynamic changes of plasma cytokine levels in patients with severe acute respiratory syndrome.Zhonghua Nei Ke Za Zhi.2003 Sep;42(9):643-645.
43 Zhang Y,Li J,Zhan Y,Wu L,Yu X,Zhang W,Ye L,Xu $,Sun R,Wang Y,Lou J.Analysis of serum cytokines in patients with severe acute respiratory syndrome.Infect Immun.2004 Aug;72(8):4410-4415.
44 张永宏 陈新月 闰惠平 檀玉芬 温韬 刁士琦 吴吴.SARS患者血清细胞因子临床意义探讨[J].中华微生物学和免疫学杂志.2004.24(3):178-180.
45 黄利坚 刘树人.传染性非典型肺炎患者外周血IFN-γ、TNF-α、IL-4、IL-10细胞因子的表达研究[J].中华医学研究杂志2004年8月第4期
46 Hogan RJ,Gao G,Rowe T,et al.Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Statl.J Virol 2004;78:11416-11421.
47 Benencia F,Courreges MC.Nitric oxide and macrophage antiviral extrinsic activity.Immunology.1999 Nov;98(3):363-370.
48 Pope M,Marsden PA,Cole E,Sloan S,Fung LS,Ning Q,Ding JW,Leibowitz JL,Phillips M J,Levy GA.Resistance to murine hepatitis virus strain 3 is dependent on production of nitric oxide.J Virol.1998 Sep;72(9):7084-7090.
49 Akerstrom S,Mousavi-Jazi M,Klingstrom J,Leijon M,Lundkvist A,Mirazimi A.Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus.J Virol.2005 Feb;79(3):1966-1969.
50 Keyaerts E,Vijgen L,Chen L,Maes P,Hedenstierna C,Van Ranst M.Inhibition of SARS-coronavirus infection in vitro by S-nitroso-N-acetylpenicillamine,a nitric oxide donor compound.Int J Infect Dis.2004 Jul;8(4):223-226.
51 吴新忠 庄俊华 陈林 黄宪章 林琳 邹国林.监测SARS病人IFN-γ、IFN-α、NO和iNOS水平及其意义[J].中国病理生理杂志.2004,20(1).-133-136.
1 World Health Organization (WHO). Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003.http://www.who.int/csr/sars/country/table2004_04_21/en/, 2003(accessed Feb 3, 2005).
2 Reilley B, Van Herp M, Sermand D, Dentico N. SARS and Carlo Urbani.N Engl J Med 2003; 348: 1951-52.
3 He JF, Xu RH, Yu DW, et al. Severe acute respiratory syndrome in Guangdong Province of China: epidemiology and control measures. Zhonghua Yu Fang Yi Xue Za Zhi. 2003; 37: 227-32.
4 Tsang KW, Ho PL, Ooi GC, et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1977 - 85.
5 Lee N, Hui D, Wu A, et al. A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1986-94.
6 Kuiken, T. Fouchier RA, Schutten M, et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome.Lancet 2003; 362: 263-70.
7 Peiris JS, Lai ST, Poon LL, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003; 361:1319-25.
8 Ksiazek TG, Erdman D, Goldsmith C, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003;348: 1953-66.
9 Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348: 1967-76.
10 Marra MA, Jones SJ, Astell CR, et al. The Genome sequence of the SARS-associated coronavirus. Science 2003; 300: 1399-404.
11 Rota PA, Oberste MS, Monroe SS, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome.Science 2003; 300: 1394-99.
12 Poon LL, Wong OK, Chan KH, et al. Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin Chem 2003; 49:953-55.
13 Yam WC, Chan KH, Poon LL, et al. Evaluation of reverse transcription-PCR assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus. J Clin Microbiol 2003; 41:4521-24.
14 Che XY, Qiu LW, Pan YX, et al. Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome. J Clin Microbiol 2004; 42:2629-35.
15 Sun HY, Fang CT, Wang JT, Chen YC, Chang SC. Treatment of severe acute respiratory syndrome in health-care workers. Lancet 2003; 362:2025-26.
16 So LK, Lau AC, Yam LY, et al. Development of a standard treatment protocol for severe acute respiratory syndrome. Lancet 2003; 361:1615-17.
17 Ward SE, Loutfy MR, Blatt LM, et al. Dynamic changes in clinical features and cytokine/chemokine responses in SARS patients treated with interferon alfacon-1 plus corticosteroids. Antivir Ther 2005; 10:263-75.
18 Cinatl J Jr, Michaelis M, Scholz M, Doerr HW. Role of interferons in the treatment of severe acute respiratory syndrome. Expert Opin Biol Ther 2004; 4: 827-36.
19 Crowe JE Jr, Bui PT, London WT, et al. Satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis. Vaccine 1994; 12:691-99.
20 Novak M, Moldoveanu Z, Schafer DP, Mestecky J, Compans RW. Murine model for evaluation of protective immunity to influenza virus. Vaccine 1993; 11:55-60.
21 Osterhaus AD, Fouchier RA, Kuiken T. The aetiology of SARS: Koch's postulates fulfilled. Philos Trans R Soc Lond B Biol Sci 2004;359:1081-82.
22 Fouchier RA, KuikenT, Schutten M, et al. Aetiology: Koch's postulates fulfilled for SARS virus. Nature 2003; 423:240.
23 Rowe T, Gao G, Hogan RJ, et al. Macaque model for severe acute respiratory syndrome. J Virol 2004; 78:11401-04.
24 McAuliffe J, Vogel L, Roberts A, et al. Replication of SARS coronavirus administered into the respiratory tract of African Green,rhesus and cynomolgus monkeys. Virology 2004; 330:8-15.
25 Qin C, Wang J, Wei Q, et al. An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus. J Pathol 2005;206:251-59.
26 Lawler JV, Endy TP, Hensley LE, et al. Cynomolgus macaque as an animal model for severe acute respiratory syndrome. PLoS Med 2006; 3:e149.
27 Greenough TC, Carville A, Coderre J, et al. Pneumonitis and multi-organ system disease in common marmosets (Callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus. Am J Pathol 2005; 167:455-63.
28 Sub'barao K, McAuliffe J, Vogel L, et al. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice.J Virol 2004; 78:3572-77.
29 Wentworth DE, Gillim-Ross L, Espina N, Bernard KA. Mice susceptible to SARS coronavirus. Emerg Infect Dis 2004; 10:1293-96.
30 Roberts A, Paddock C, Vogel L, et al. Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans. J Virol 2005; 79:5833-38.
31 Glass WG, Subbarao K, Murphy B, Murphy PM. Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 2004; 173:4030-39.
32 Hogan RJ, Gao G, Rowe T, et al. Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Statl. J Virol 2004; 78:11416-21.
33 Roberts A, Vogel L, Guarner J, et al. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters. J Virol 2005; 79:503-11.
34 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425:915.
35 Wu D, Tu C, Xin C, et al. Civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates. J Virol 2005; 79:2620-25.
36 Haagmans BL, Kuiken T, Martina BE, et al. Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques. Nat Med 2004; 10:290-93.
37 Gao W, Tamin A, Soloff A, et al. Effects of a SARS-associated coronavirus vaccine in monkeys. Lancet 2003; 362:1895-96.
38 Bukreyev A, Lamirande EW, Buchholz UJ, et al. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 2004; 363:2122-27.
39 Yang ZY, Kong WP, Huang Y, et al. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 2004; 428:561-64.
40 Bisht H, Roberts A, Vogel L, et al. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc Natl Acad Sci U S A 2004;101:6641-46
41 Greenough TC, Babcock GJ, Roberts A, et al. Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice. J Infect Dis 2005; 191:507-14.
42 Roberts A, Thomas WD, Guarner J, et al. Therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden Syrian hamsters. J Infect Dis 2006;193:685-92.
43 Sui J, Li W, Roberts A, et al. Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants. J Virol 2005; 79:5900-06.
44 ter Meulen J, Bakker AB, van den Brink EN, et al. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.Lancet 2004; 363:2139-41.
45 Weingartl H, Czub M, Czub S, et al. Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets.J Virol 2004; 78:12672-76.
46 Li BJ, Tang Q, Cheng D, et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat Med 2005; 11:944-51.
47 Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003;426:450-54.
48 http://www. hms.harvard. edu/dms/bbs/fac/farzanmir. html
49 http://research.utmb. edu/ctd/Tech_new. asp
50 Tseng CT, Huang C, Newman P, et al. SARS Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 (hACE2) Virus Receptor [J]. J Virol, 2006, Nov 15; [Epub ahead of print]
51 Donoghue M, Wakimoto H, Maguire CT, et al. Heart block, ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins [J]. J Mol Cell Cardiol, 2003, 35:1043-1053.
52 McCray PB Jr, Pewe L, Wohlford-Lenane C, et al. LETHAL INFECTION IN K18-hACE2 MICE INFECTED WITH SARS-CoV [J]. J Virol, 2006, Nov 1; [Epub ahead of print]
53 Subbarao K, Roberts A. Is there an ideal animal model for SARS? Trends Microbiol 2006; 14:299-303.
54 Peiris JS, Guan Y, Yuen KY. Severe acute respiratory syndrome. Nat Med 2004; 10:S88-97
55 http://www. ealing. gov. uk/services/environment/pest_control/types_of_pest/mice/
56 http://media. nasaexplores. com/lessons/03-026/images/microscope.jpg
57 http://www. globalhealth. org/view_top. php3?id=228
58 http://www. ahc. umn. edu/rar/MNAALAS/Research_overview. pdf#search=%22Research overview. pdf%22)
2 Kuiken, T. Fouchier RA, Schutten M, et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 2003; 362:263-270.
3 Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348:1967-1976.
4 Tsang KW, Ho PL, Ooi GC, et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1977-1985.
5 He JF, Xu RH, Yu DW, et al. Severe acute respiratory syndrome in Guangdong Province of China: epidemiology and control measures.Zhonghua Yu Fang Yi Xue Za Zhi. 2003; 37: 227-232.
6 Bukreyev A, Lamirande EW, Buchholz UJ, et al. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 2004; 363:2122-2127.
7 Lawler JV, Endy TP, Hensley LE, et al. Cynomolgus macaque as an animal model for severe acute respiratory syndrome. PLoS Med 2006; 3:el49.
8 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425:915.
9 Qin C, Wang J, Wei Q, et al. An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus. J Pathol 2005;206:251-259.
10 Roberts A, Paddock C, Vogel L, et al. Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans. J Virol 2005; 79:5833-5838.
11 Roberts A, Vogel L, Guarner J, et al. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters. J Virol 2005; 79:503-511
12 Rowe T, Gao G, Hogan RJ, et al. Macaque model for severe acute respiratory syndrome. J Virol 2004; 78:11401 -11404.
13 Babcock GJ, Esshaki DJ, Jr Thomas WD, et al Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol 2004; 78:4552-4560.
14 Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003; 426:450-454.
15 Wang P, Chen J, Zheng A, et al. Expression cloning of functional receptor used by SARS coronavirus. Biochem Biophys Res Commun 2004;315:439-444.
16 McCray PB Jr, Pewe L, Wohlford-Lenane C, et al. LETHAL INFECTION IN K18-hACE2 MICE INFECTED WITH SARS-CoV. J Virol 2007;81:813-821.
17 Tseng CT, Huang C, Newman P, et al. SARS Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 (hACE2) Virus Receptor.J Virol 2007; 81:1162-1173.
18 Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG,Birnberg NC, Evans RM. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature.1982 Dec 16; 300 (5893):611-615.
19 Baeumler S, Wulff D, Tagliani L, Song P. A real-time quantitative PCR detection method specific to widestrike transgenic cotton (event 281-24-236/3006-210-23). J Agric Food Chem. 2006 Sep 6;54(18):6527-6534.
20 Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB,Mourrain P, Palauqui JC, Vernhettes S. Transgene-induced gene silencing in plants. Plant J. 1998 Dec;16 (6):651-659.
21 Hamming, Timens W, Bulthuis MLC, et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004; 203:631-637.
22 Crackower MA, Sarao R, Oudit GY, et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 2002; 417:822-828.
23 Donoghue M, Hsieh F, Baronas E, et al. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res 2000; 87:E1-E9.
24 Mary Donoghue, Hiroko Wakimoto, Colin T Maguire, et al. Heart block,ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins. J Mol Cell Cardiol. 2003 Sep ;35 (9): 1043-1053
25 Subbarao K, McAuliffe J, Vogel L, et al. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J Virol 2004;78:3572-3577.
26 Leung TF, Wong GW, Hon KL, Fok TF. Severe acute respiratory syndrome (SARS) in children: epidemiology, presentation and management. Paediatr Respir Rev. 2003 Dec; 4 (4):334-339.
27 Rainer TH, Chan PK, Ip M, Lee N, Hui DS, Smit D, Wu A, Ahuja AT, Tarn JS,Sung JJ, Cameron P. The spectrum of severe acute respiratory syndrome-associated coronavirus infection. Ann Intern Med. 2004 Apr 20;140 (8):614-619.
28 Osterhaus AD, Fouchier RA, Kuiken T. The aetiology of SARS: Koch's postulates fulfilled. Philos Trans R Soc Lond B Biol Sci 2004; 359:1081-82.
29 Fouchier RA, Kuiken T, Schutten M, et al. Aetiology: Koch's postulates fulfilled for SARS virus. Nature 2003; 423:240.
30 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425:915.
31 Wentworth DE, Gillim-Ross L, Espina N, Bernard KA. Mice susceptible to SARS coronavirus. Emerg Infect Dis 2004; 10:1293-1296.
32 Glass WG, Subbarao K, Murphy B, Murphy PM. Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 2004;173:4030-4039.
33 Hwang DM, Chamberlain DW, Poutanen SM, et al. Pulmonary pathology of severe acute respiratory syndrome in Toronto. Mod Pathol 2005; 18:1-10.
34 Lang ZW, Zhang LJ, Zhang SJ, et al. A clinicopathological study of three cases of severe acute respiratory syndrome (SARS). Pathology 2003;35:526-531.
35 Ding Y, Wang H, Shen H, et al. The clinical pathology of severe acute respiratory syndrome(SARS):a report from China.d Pathol 2003;200:282-289.
36 Jeffers,S.A.,Tusell,S.M.,Gillim-Ross,L.,Hemmila,E.M.,Achenbach,J.E.,Babcock,G.J.,Thomas,W.D.Jr.,Thackray,L.B.,Young,M.D.,Mason,R.J.,Ambrosino,D.M.,Wentworth,D.E.,DeMartini,J.C.,Holmes,K.V.(2004).CD209L(L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus.Proc.Natl.Acad.Sci.U.S.A.101:15748-15753.
37 石玉玲 李林海 徐德兴 等.广东地区严重急性呼吸综合征(SARS)患者血清IFA检测[J].细胞与分子免疫学杂志,2003,19(5):封2.
38 石玉玲 李林海 石凌波 张亚松 徐德兴万根平顾晓琼 张小玲.广州地区住院患儿血清抗SARS病毒抗体的检测[J].细胞与分子免疫学杂志,2003年19卷6期
39 崔小岱 宋国维 马连华 许峥 王天有.儿童SARS病毒抗体回顾检测及其意义[J].中华医学研究杂志,2004年第4卷第3期
40 Nicholls,J.M.,Lee,K.C.,Lee,K.C.(2003).Lung pathology of fatal severe acute respiratory syndrome.LANCET 361:P 1773-1778.
41 Wong CK,Lam CW,Wu AK,Ip WK,Lee NL,Chan IH,Lit LC,Hui DS,Chan MH,Chung SS,Sung JJ.Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome.Clin Exp Immunol.2004Apr;136(1):95-103.
42 Xie J,Han Y,Li TS,Qiu ZF,Ma X J,Fan HW,Lu W,Liu ZY,Wang Z,Wang HL,Deng GH.Dynamic changes of plasma cytokine levels in patients with severe acute respiratory syndrome.Zhonghua Nei Ke Za Zhi.2003 Sep;42(9):643-645.
43 Zhang Y,Li J,Zhan Y,Wu L,Yu X,Zhang W,Ye L,Xu $,Sun R,Wang Y,Lou J.Analysis of serum cytokines in patients with severe acute respiratory syndrome.Infect Immun.2004 Aug;72(8):4410-4415.
44 张永宏 陈新月 闰惠平 檀玉芬 温韬 刁士琦 吴吴.SARS患者血清细胞因子临床意义探讨[J].中华微生物学和免疫学杂志.2004.24(3):178-180.
45 黄利坚 刘树人.传染性非典型肺炎患者外周血IFN-γ、TNF-α、IL-4、IL-10细胞因子的表达研究[J].中华医学研究杂志2004年8月第4期
46 Hogan RJ,Gao G,Rowe T,et al.Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Statl.J Virol 2004;78:11416-11421.
47 Benencia F,Courreges MC.Nitric oxide and macrophage antiviral extrinsic activity.Immunology.1999 Nov;98(3):363-370.
48 Pope M,Marsden PA,Cole E,Sloan S,Fung LS,Ning Q,Ding JW,Leibowitz JL,Phillips M J,Levy GA.Resistance to murine hepatitis virus strain 3 is dependent on production of nitric oxide.J Virol.1998 Sep;72(9):7084-7090.
49 Akerstrom S,Mousavi-Jazi M,Klingstrom J,Leijon M,Lundkvist A,Mirazimi A.Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus.J Virol.2005 Feb;79(3):1966-1969.
50 Keyaerts E,Vijgen L,Chen L,Maes P,Hedenstierna C,Van Ranst M.Inhibition of SARS-coronavirus infection in vitro by S-nitroso-N-acetylpenicillamine,a nitric oxide donor compound.Int J Infect Dis.2004 Jul;8(4):223-226.
51 吴新忠 庄俊华 陈林 黄宪章 林琳 邹国林.监测SARS病人IFN-γ、IFN-α、NO和iNOS水平及其意义[J].中国病理生理杂志.2004,20(1).-133-136.
1 World Health Organization (WHO). Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003.http://www.who.int/csr/sars/country/table2004_04_21/en/, 2003(accessed Feb 3, 2005).
2 Reilley B, Van Herp M, Sermand D, Dentico N. SARS and Carlo Urbani.N Engl J Med 2003; 348: 1951-52.
3 He JF, Xu RH, Yu DW, et al. Severe acute respiratory syndrome in Guangdong Province of China: epidemiology and control measures. Zhonghua Yu Fang Yi Xue Za Zhi. 2003; 37: 227-32.
4 Tsang KW, Ho PL, Ooi GC, et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1977 - 85.
5 Lee N, Hui D, Wu A, et al. A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1986-94.
6 Kuiken, T. Fouchier RA, Schutten M, et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome.Lancet 2003; 362: 263-70.
7 Peiris JS, Lai ST, Poon LL, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003; 361:1319-25.
8 Ksiazek TG, Erdman D, Goldsmith C, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003;348: 1953-66.
9 Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348: 1967-76.
10 Marra MA, Jones SJ, Astell CR, et al. The Genome sequence of the SARS-associated coronavirus. Science 2003; 300: 1399-404.
11 Rota PA, Oberste MS, Monroe SS, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome.Science 2003; 300: 1394-99.
12 Poon LL, Wong OK, Chan KH, et al. Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin Chem 2003; 49:953-55.
13 Yam WC, Chan KH, Poon LL, et al. Evaluation of reverse transcription-PCR assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus. J Clin Microbiol 2003; 41:4521-24.
14 Che XY, Qiu LW, Pan YX, et al. Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome. J Clin Microbiol 2004; 42:2629-35.
15 Sun HY, Fang CT, Wang JT, Chen YC, Chang SC. Treatment of severe acute respiratory syndrome in health-care workers. Lancet 2003; 362:2025-26.
16 So LK, Lau AC, Yam LY, et al. Development of a standard treatment protocol for severe acute respiratory syndrome. Lancet 2003; 361:1615-17.
17 Ward SE, Loutfy MR, Blatt LM, et al. Dynamic changes in clinical features and cytokine/chemokine responses in SARS patients treated with interferon alfacon-1 plus corticosteroids. Antivir Ther 2005; 10:263-75.
18 Cinatl J Jr, Michaelis M, Scholz M, Doerr HW. Role of interferons in the treatment of severe acute respiratory syndrome. Expert Opin Biol Ther 2004; 4: 827-36.
19 Crowe JE Jr, Bui PT, London WT, et al. Satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis. Vaccine 1994; 12:691-99.
20 Novak M, Moldoveanu Z, Schafer DP, Mestecky J, Compans RW. Murine model for evaluation of protective immunity to influenza virus. Vaccine 1993; 11:55-60.
21 Osterhaus AD, Fouchier RA, Kuiken T. The aetiology of SARS: Koch's postulates fulfilled. Philos Trans R Soc Lond B Biol Sci 2004;359:1081-82.
22 Fouchier RA, KuikenT, Schutten M, et al. Aetiology: Koch's postulates fulfilled for SARS virus. Nature 2003; 423:240.
23 Rowe T, Gao G, Hogan RJ, et al. Macaque model for severe acute respiratory syndrome. J Virol 2004; 78:11401-04.
24 McAuliffe J, Vogel L, Roberts A, et al. Replication of SARS coronavirus administered into the respiratory tract of African Green,rhesus and cynomolgus monkeys. Virology 2004; 330:8-15.
25 Qin C, Wang J, Wei Q, et al. An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus. J Pathol 2005;206:251-59.
26 Lawler JV, Endy TP, Hensley LE, et al. Cynomolgus macaque as an animal model for severe acute respiratory syndrome. PLoS Med 2006; 3:e149.
27 Greenough TC, Carville A, Coderre J, et al. Pneumonitis and multi-organ system disease in common marmosets (Callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus. Am J Pathol 2005; 167:455-63.
28 Sub'barao K, McAuliffe J, Vogel L, et al. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice.J Virol 2004; 78:3572-77.
29 Wentworth DE, Gillim-Ross L, Espina N, Bernard KA. Mice susceptible to SARS coronavirus. Emerg Infect Dis 2004; 10:1293-96.
30 Roberts A, Paddock C, Vogel L, et al. Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans. J Virol 2005; 79:5833-38.
31 Glass WG, Subbarao K, Murphy B, Murphy PM. Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 2004; 173:4030-39.
32 Hogan RJ, Gao G, Rowe T, et al. Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Statl. J Virol 2004; 78:11416-21.
33 Roberts A, Vogel L, Guarner J, et al. Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters. J Virol 2005; 79:503-11.
34 Martina BE, Haagmans BL, Kuiken T, et al. Virology: SARS virus infection of cats and ferrets. Nature 2003; 425:915.
35 Wu D, Tu C, Xin C, et al. Civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates. J Virol 2005; 79:2620-25.
36 Haagmans BL, Kuiken T, Martina BE, et al. Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques. Nat Med 2004; 10:290-93.
37 Gao W, Tamin A, Soloff A, et al. Effects of a SARS-associated coronavirus vaccine in monkeys. Lancet 2003; 362:1895-96.
38 Bukreyev A, Lamirande EW, Buchholz UJ, et al. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 2004; 363:2122-27.
39 Yang ZY, Kong WP, Huang Y, et al. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 2004; 428:561-64.
40 Bisht H, Roberts A, Vogel L, et al. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc Natl Acad Sci U S A 2004;101:6641-46
41 Greenough TC, Babcock GJ, Roberts A, et al. Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice. J Infect Dis 2005; 191:507-14.
42 Roberts A, Thomas WD, Guarner J, et al. Therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden Syrian hamsters. J Infect Dis 2006;193:685-92.
43 Sui J, Li W, Roberts A, et al. Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants. J Virol 2005; 79:5900-06.
44 ter Meulen J, Bakker AB, van den Brink EN, et al. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.Lancet 2004; 363:2139-41.
45 Weingartl H, Czub M, Czub S, et al. Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets.J Virol 2004; 78:12672-76.
46 Li BJ, Tang Q, Cheng D, et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat Med 2005; 11:944-51.
47 Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003;426:450-54.
48 http://www. hms.harvard. edu/dms/bbs/fac/farzanmir. html
49 http://research.utmb. edu/ctd/Tech_new. asp
50 Tseng CT, Huang C, Newman P, et al. SARS Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 (hACE2) Virus Receptor [J]. J Virol, 2006, Nov 15; [Epub ahead of print]
51 Donoghue M, Wakimoto H, Maguire CT, et al. Heart block, ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins [J]. J Mol Cell Cardiol, 2003, 35:1043-1053.
52 McCray PB Jr, Pewe L, Wohlford-Lenane C, et al. LETHAL INFECTION IN K18-hACE2 MICE INFECTED WITH SARS-CoV [J]. J Virol, 2006, Nov 1; [Epub ahead of print]
53 Subbarao K, Roberts A. Is there an ideal animal model for SARS? Trends Microbiol 2006; 14:299-303.
54 Peiris JS, Guan Y, Yuen KY. Severe acute respiratory syndrome. Nat Med 2004; 10:S88-97
55 http://www. ealing. gov. uk/services/environment/pest_control/types_of_pest/mice/
56 http://media. nasaexplores. com/lessons/03-026/images/microscope.jpg
57 http://www. globalhealth. org/view_top. php3?id=228
58 http://www. ahc. umn. edu/rar/MNAALAS/Research_overview. pdf#search=%22Research overview. pdf%22)
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