Susceptibility Of HACE2 Transgenic Mice To SARS-CoV And Experimental Study On SARS Pathogenesis

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.