Construction Of Recombinant Vesicular Stomatitis Virus Expressing Marburg Virus Glycoprotein And The Establishment And Application Of Animal Model

Marburg virus disease（MVD）is an acute febrile and virulent infectious disease with severe hemorrhage caused by Marburg virus（MARV）.Since the first outbreak in1967,MVD has been sporadically distributed mainly in central Africa,with a mortality rate of 90%,posing a serious threat to human life,and there is no approved vaccine and therapeutic countermeasures for MVD.The most recent MVD outbreak occurred in February 2023 in Guinea,caused at least 9 deaths,and the attention on MARV has increased significantly both domestically and internationally,and the corresponding research on MARV vaccines and antibodies is advancing in an orderly manner.MARV should be handled in biosafety level 4（BSL-4）laboratory,which hampered the development and further evaluation of prophylactic and therapeutic approaches against MARV.Vesicular stomatitis virus（VSV）reverse genetic technology has been widely used for recombinant viruses,which are safe and easy to package exogenous glycoproteins on the virus surface,and VSV recombinant（r VSV）can mimicking the original virus for neutralizing antibody detection.In addition,it has been shown that high titers of MARV recombinant VSV infecting Syrian hamsters cause death and have the potential to be an alternative animal model for MARV.The aim of this study was to construct a surrogate virus for neutralizing antibody testing in a biosafety level 2（BSL-2）laboratory and a surrogate animal model for evaluating antibodies and vaccines targeting MARV GP.This study consists of three main parts.1.Construction and identification of MARV GP recombinant VSV and its application in neutralizing antibody detection.Three recombinant full-length plasmids were obtained by replacing the G genes in the full-length plasmids of VSV infectious clones carrying the e GFP tag with the GP genes of the three MARVs.Three recombinant full-length plasmids were cotransfected with four VSV helper plasmids,respectively,to rescue three MARV GP recombinant VSV viruses carrying e GFP tags（VSV-Angola,VSV-Musoke,VSV-Ravn）.Western blot（WB）,indirect immunofluorescence（IF）and reverse transcription polymerase chain reaction（RT-PCR）were used to identify the recombinant virus;Viral titers and growth kinetic of recombinant virus were measured.Then the recombinant viruses were applied to neutralizing antibody detection assays.The results showed that the three replication-competent MARV GP recombinant VSVs carrying e GFP tags were successfully rescued and applied to antibody neutralization potency assays.The neutralization titer of MR191 was 1:20 and that of MR78 was 1:160.2.Establishment of VSV-Angola infection model in Syrian hamsters.The three recombinant viruses were infected intraperitoneally with 10^6.5TCID₅₀in4～8 weeks old Syrian hamsters to observe their clinical characteristics;to evaluate the safety of VSV-Angola in BALB/c mice,SD rats and guinea pigs;to determine the LD₅₀of VSV-Angola infected Syrian hamsters;Detection of viral load in each organ,pathological changes in each organ,routine blood and blood biochemical changes after VSV-Angola infection in Syrian hamster.Protection against challenge(infection with50-fold LD₅₀of VSV Angola)was assessed 1 week after immunization of golden hamsters with VSV-Musoke,VSV-Ravn.The results showed that only VSV-Angola among the three recombinant viruses was lethal to Syrian hamster,and it was found that VSV-Angola infected Syrian hamster had extensive tissue tropism and obvious hepatotropism,and caused severe liver failure,exhibiting symptoms similar to those of MARV infection in non-human primates.Successfully established a VSV-Angola Syrian hamster lethal infection model and applied it to evaluate the protective effect of MARV vaccine.3.Exploring the effect of MARV RBS structural domain on the pathogenicity of VSV-Angola.The sequences of the RBS structural domain in the three MARV GPs,Angola-GP,Musoke-GP,and Ravn-GP,were comparatively analyzed using Meg Align software to obtain the specific amino acid of the RBS structural domain in Angola-GP,that is,the amino acid at position 74 of the GP.By point mutation technique,the 74A（alanine）in Angola-GP was mutated to 74T（threonine）,then the full-length plasmid of Angola-GP74T recombinant VSV was constructed,and the mutated recombinant virus VSV-Angola74T was rescued by VSV reverse genetic technique.VSV-Angola74T was infected intraperitoneally with 106.5 TCID₅₀to investigate whether there was any change in its pathogenicity to Syrian hamsters.The results showed that the lethality of VSV-Angola74T was consistent with VSV-Angola after infection of Syrian hamsters,indicating that the mutation of amino acid 74th position in GP did not significantly affect the pathogenicity of VSV-Angola,meaning that the RBS structural domain of GP did not significantly affect the virulence of VSV-Angola.In this study,three different MARV GP recombinant VSVs were constructed based on the VSV reverse genetic system and successfully applied to neutralizing antibody detection.We also established a model of Syrian hamster infection based on recombinant VSV-Angola,which can be applied to the evaluation and primary screening of MARV vaccine and antibody.Finally,the insignificant effect of the RBS structural domain on the pathogenicity of VSV-Angola was explored and verified by point mutation and VSV reverse genetic techniques in combination with the Syrian hamster infection model.