Application And Evaluation Of The Animal Nose-only Aerosol Exposure Device In Pathogen Infectious Animal Models

Objective To conduct performace evaluation of the animal nose-only exposure device and to verify sterilization effect,which provides security guarantee for biosafety experiments and infectious activities.Methods Escherichia coli(E.coli)aerosol and mouse hepatitis virus(MHV)aerosol without pathogenicity to human and selected to represent bacteria and viruses respectively as model microorganisms were generated by the nose-only inhalation exposure device,which evaluated the effect of aerosol generation and activiy.And a solution of 75%ethanol and 3%hydrogen peroxide was run through the nebulizer to disinfect the system,which evaluated the effect of sterilization.E.coli and MHV were collected respectively in the exposure chamber,at sites where leaking easily occurred,including after disinfection.The sampled E.coli was cultured to count colony.In addition,RNA from the sampled MHV was extracted and Real-time quantitative PCR was then used to test the presence of viral RNA from the samples.Results The result showed that the mass median aerodynamic diameter of the E.coli aerosol was 1.27±0.61 μm,that colonies grew on smearing plate with sample liquid collected in the exposure chamber,that the cultured colony counts by NC membrane sampling of different ports were no significant difference,and that no colony growth was found on the sedimentation plate at sites where leaking readily occurred or on the sampling plates after disinfection.There was a positive result for MHV RNA of the sampling solution from the liquid impinger,and no virus RNA was detected in the sampling solutions from the sites where leaking easily occurred and after disinfection.Conclusions The results show that the animal nose-only inhalation exposure device can operate normally with good air tightness and 100%disinfected effect,and the aerosol particles 1.27 ±0.61 μm generated by the device pass through and distribute evenly in each port of the exposure chamber during operation,which provides the basis for the safe,effective application of the device in animal infection of highly pathogenic microorganisms.Objective To simulate the natural infection route,and to establish the animal model infected by influenza A H1N1 virus aerosol device,compared with the intranasal infectious mice models,which provides the experimental basis for the application of mice model infected with H1N1 aerosol device.Methods The BALB/C mice were randomly divided 3 groups:aerosol group,intranasal group and control group.Mice in aerosol group and intranasal group were respectively challenged with H1N1 virus by aerosol inhalation and intranasal instillation,and mice were exposure with containing-MEM aerosol inhalation infection as control group.We compared the weight changes and survival rates of the mice for 14 consecutive days after inoculation,The mice were euthanized at days 3,5,7,and 9 post-infection to perform necropsies,to conduct pathological analyses of the lungs,and to measure viral titers in the tissues.Results The mice in the aerosol group showed clinical symptoms on day 4,obvious lung lesions on day 5,rapid weight loss on day 7 and peak virus replication of lungs on days 7 to 9.However,the mice in the instillation group post-inoculation exhibited clinical signs on day 2,rapid weight loss and obvious lung lesions on day 3,and virus replication of lungs peaked on days 3 to 5.Bronchial epithelial hyperplasia was detected in lungs post-exposure,but no detected in instillation group.The disease and lung lesion progress in the mice that inhaled H1N1-containing aerosol were slower than those of instillation-inoculated mice,and lung lesions were homogeneous in the aerosol mice and heterogeneous in the intranasal mice.Conclusions BALB/C mouse models infected by H1N1 aerosol device were successfully established and compared with intranasal mouse models,and the infectious route and the characteristics of the lung pathology in aerosol mouse models as a supplement to intransal mouse models more closely resembled those observed in humans,which provides a reference for the research mechanism of influenza development,the evaluation of relevant drug and vaccine.Objective Transgenice hDPP4 mice were infected with MERS-CoV by aerosol and instillation routes,and to compare their characteristics,which provides the experimental basis for the application and study of MERS-CoV mouse models established by aerosol exposure device.Methods Transgenic mice were randomly divided into an aerosol group,an instillation group,an aerosol control group and an instillation control group.Mice in aerosol and instillation group were respectively infected with MERS Coronavirus by aerosol inhalation and intranasal instillation routes,and mice in aerosol and instillation control groups were respectively challenged with MEM by aerosol inhalation and intranasal instillation routes as control groups.We compared the weight change and survival rates of the mice for 14 consecutive days after inoculation.The mice were necropsied at days 3,5,7,and 9 post infection to conduct pathological and immunohistochemical analyzes,and to measure virus load and cytokine levels of tissues.Results In the aerosol group,inoculation period of disease was 5-7 days.The mice after infection showed obvious lung lesions on day 7,peak virus replication of lungs on days 7 to 9 and continuous rapid weight loss on days 7-11,and gradually progressed to moderate diffuse pneumonia and even death.However,the mice in the instillation group post-inoculation exhibited clinical signs on day 1,obvious lung lesions on day 3,continuous weight loss and 0%survival rate within day 5,and virus replication of lungs peaked on days 3 to 5.Virus antigens were detected in the lungs of mice of MERS-CoV infection both by aerosol and instillation routes.The progressions of disease,lung lesion and virus replication were slower in the mice that inhaled MERS-CoV-containing aerosol than those of instillation-inoculated mice.Conclusions In this study,hDPP4 transgenic mice were successfully infected with MERS-CoV by animal nose-only exposure device,and aerosol and instillation mice all showed the clinical symptoms of acute diffuse pneumonia in humans.The route of aerosol infection in mice was consistent with that of clinical aerosol transmission,and the disease and pulmonary lesion progressions of the mice exposed to MERS-CoV aerosol more resemble with those in humans.