| Outbreaks of the emerging infectious diseases have been considered a great threat to public health.The outbreak of Ebola virus(EBOV)haemorrhagic fever in 2014,which widely spread in West African countries such as Guinea,Liberia and Sierra Leone,has attracted worldwide attention.The mortality rate of Marburg virus infection which belongs to the same filovirus family as EBOV is as high as 90%.In 2012,the Middle East respiratory syndrome(MERS),an emerging viral repiratory disease caused by a novel coronavirus(MERS-Co V),has been input to a number of countries,including China.The prevention and treatment of Lassa fever virus(LASV),which spreads mainly by rodents and has the risk of mass scale spread,is very urgent.Until now,the etiology and pathogenesis of these viruses have not been fully understood.Meanwhile,the process of drug discovery and vaccine research against these viruses is limited by the requirements of high level biosafety and the lack of easy-to-use and effective animal models.Therefore,the aim of this study is to construct high-titer pseudovirus and eatablish live imaging mouse models to partially resolve the above problems.Previously,we have modified backbone plasmid pSG3?env by insertion of a firefly luciferase(Fluc)gene with a high-efficicient promotor to increase the titer of pseudovirus.In this study,we optimized the factors which influence the titer of pseudovirus,including packaging cell,expression plasmid,backbone plasmid,the proportion of plasmid,transfection reagent.With the optimization,the titer of EBOV,MARV,MERS-CoV,LASV were increased by 100-1000 folds.In this study,a series of safe and high-throughput neutralizing antibody assays were established by using these non-replicating pseudovirus.The key factors of these assays were optimized inculding cell tropism,detection time,cell amount,virus dose and so on.These methods are sensitive,specific and reproducible,and can be used for high-throughput screening and potency evaluation of antiviral agents such as vaccines,monoclonal antibodies and small molecule compounds.Based on the high titer pseudovirus,a series of pseudovirus infection mice models were established including EBOV,MARV,MERS-CoV,LASV by optimizing the pseudotyped virus infected mice strains,weeks,infectious route,detection time,etc.With these visualized mice model,the protection efficacy of vaccines,antibodies,inhibitor agents could be evaluated against these high-pathogenic viruses in vivo.The titer of EBOV pseudovirus prepared in this study can reached 1×107 TCID50/mL.The sensitive cell line was determined as 293 T.The neutralizing activity of 4 murine mAb were detected in vitro and in vivo.The M318 and M501 showed relatively high potency.The titer of MARV pseudovirus was 9.75×107 TCID50/m L,and the sensitive cell for which was identified as 293 T.Sera obtained from guinea pigs which were immunized with pseudovirus have neutralizing activity.In the mouse model,the serum can protect the mice from virus infection.The titer of MERS-CoV pseudovirus was 1.27×108 TCID50/mL,for which the sensitive cell line was Huh 7,and the mouse strain was DPP4 KI model animal.It was proved that mAb H111-1 and R723-NEU have high neutralizing activity and have the protective effects both in vivo and in vitro.The titer of LASV pseudovirus was 1.67×108 TCID50/mL.The sensitive cell line was 293 T.Mice immunized with the DNA vaccine and the pseudovirus vaccine could be protected from pseudovirus infection.In this study,a series of safe,sensitive and specific antiviral activity evalution cytologic methods and visualized mice model have been successfully established and optimized.The platform can be operated in BSL-2 laboratory which solve the technical bottleneck of pathogen and product evaluation of highly pathogenic infectious diseases.And it provided an ideal detection technique for high-throughput screening and potency evaluation of antiviral agents such as drugs,vaccines and small molecule compounds. |
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