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1、Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice Marta L. DeDiego a, Lecia Peweb, Enrique Alvareza, Maria Teresa Rejasc, Stanley Perlmanb, Luis Enjuanesa, a Department of Molecular and Cell Biology, Centro Nacional de Biotecnologa (CSIC), Campus Univer
2、sidad Autnoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain b Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA c Centro de Biologa Molecular (CSIC-UAM), Facultad de Ciencias, Campus Universidad Autnoma, Cantoblanco, 28049 Madrid, Spain a r t i c l ei n f oa b s t r a c t Arti
3、cle history: Received 17 January 2008 Returnedtoauthorforrevision21February2008 Accepted 10 March 2008 Available online 2 May 2008 Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specifi c genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-69b), the structural gene E (rSARS-C
4、oV-E), and a combination of both sets of genes (rSARS-CoV-E,69b) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.1 and CaCo-2) cell lines and for transgenic (Tg) mice expressing the SARS-CoV receptor human angiotensin co
5、nverting enzyme-2 (hACE-2), indicating that none of these proteins is essential for the viral cycle. Furthermore, in Vero E6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the fi nal morph
6、ology of the virions. Nevertheless, in the absence of E protein, release of virus particles effi cacy was reduced. Viruses lacking E protein grew about 100-fold lower than the wt virus in lungs of Tg infected mice but did not grow in the brains of the same animals, in contrast to the rSARS-CoV-69b v
7、irus, which grew almost as well as the wt in both tissues. Viruses lacking E proteinwere highly attenuated in the highlysensitive hACE-2 Tg mice, in contrast tothe minimal rSARS-CoV-69b and wt viruses. These data indicate that E gene might be a virulence factor infl uencing replication level, tissue
8、 tropism and pathogenicity of SARS-CoV, suggesting that E attenuated viruses are promising vaccine candidates. 2008 Elsevier Inc. All rights reserved. Keywords: Coronavirus SARS-CoV hACE-2 transgenic mice Introduction The etiologic agent causing severe acute respiratory syndrome (SARS) is a novel co
9、ronavirus (CoV) named SARS-CoV (Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Marra et al., 2003; Peiris et al., 2003; Rota et al., 2003). The disease, which caused an average mortality of approximately 10% and for which no defi ned therapy is available, was
10、 reported for the fi rst time in Guandong province, China, at the end of 2002, and rapidlyspread to 32 countries. After July 2003, only four community acquired cases were reported in China, although there have been three instances of laboratory-acquired infections described (http:/www.who.int/csr/ s
11、ars/en/). Initial investigations indicated that SARS-CoV spread to humans from infected wild animals in wet markets of Southern China, such as Himalayan palm civets (Paguna larvatta) and Chinese ferret badgers (Melogale moschatta) (Guan et al., 2003). Nevertheless, the recognition of SARS-like CoVs
12、in bats suggests that these species are most likely the natural reservoir of SARS-CoV (Lau et al., 2005; Li et al., 2005). Therefore, the reemergence of the virus remains a possibility and the engineering of attenuated viruses as research tools and vaccine can- didates is of high interest. SARS-CoV
13、is an enveloped virus of the Coronaviridae family, and hasa single-stranded, positive sense29.7 kb RNAgenome (Gorbalenya et al., 2004; Snijder et al., 2003). Human coronaviruses have been divided into different groups (Enjuanes et al., 2008b). Group 1 in- cludes the human coronavirus 229E (HCoV-229E
14、), generally asso- ciated with the common cold, and HCoV-NL63, which causes more severe lower respiratory diseases (Fouchier et al., 2004; Kaiser et al., 2005; van der Hoek et al., 2004). Group 2 human CoVs include HCoV- OC43, which has been associated with common colds, the recently described HCoV-
15、HKU1, which was identifi ed in adults with pneumo- nia (Woo et al., 2005), and SARS-CoV. Among human CoVs, SARS-CoV causes the most severe disease (Weiss and Navas-Martin, 2005). Coronaviruses replicate in the cell cytoplasm and encode a nested set of mRNA molecules of different sizes. Viral genome
16、expression begins with the translation of two large polyproteins, pp1a and pp1ab, including the viral replicase genes (Thiel et al., 2003). These genes are involved in genome replication and transcription of subgenomic mRNAs (sg mRNAs), encoding structural proteins such as the spike (S), envelope (E), membrane (M), andnucleocapsid (N),anda setofgroup- specifi c proteins, whose sequence and number differs among the different species of coronavirus (Enjuanes et al., 2008b). In the case of SARS-C
