Monitoring Neutralization Property Change of Evolving Hantaan and Seoul Viruses with a Novel Pseudovirus-Based Assay

The Hantaan virus(HTNV)and Seoul virus(SEOV)mutants have accumulated over time.It is important to determine whether their neutralizing epitopes have evolved,thereby making the current vaccine powerless.However,it is impossible to determine by using traditional plaque reduction neutralization test(PRNT),because it requires large numbers of live mutant strains.Pseudovirus-based neutralization assays(PBNA)were developed by employing vesicular stomatitis virus(VSV)backbone incorporated with HTNV or SEOV glycoproteins(VSVDG*-HTNVG or VSVDG*-SEOVG).56 and 51 single amino acid substitutions of glycoprotein(GP)in HTNV and SEOV were selected and introduced into the reference plasmid.Then the mutant pseudoviruses were generated and tested by PBNA.The PBNA results were highly correlated with PRNT ones with R2 being 0.91 for VSVDG*-HTNVG and 0.82 for VSVDG*-SEOVG.53 HTNV mutant pseudoviruses and 46 SEOV mutants were successfully generated.Importantly,by using PBNA,we found that HTNV or SEOV immunized antisera could neutralize all the corresponding 53 HTNV mutants or the 46 SEOV mutants respectively.The novel PBNA enables us to closely monitor the effectiveness of vaccines against large numbers of evolving HTNV and SEOV.And the current vaccine remains to be effective for the naturally occurring mutants.

Development and effectiveness of pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro

With the development of the COVID-19 epidemic,there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2(BSL-2)facilities.Previously,researchers had developed a pseudotyped virus systemfor SARS-CoV andMERS-CoV,based onHIV-1 core,bearing virus spike protein.During the development of a pseudotyped SARS-CoV-2 system,a eukaryotic expression plasmid expressing SARSCoV-2 spike(S)protein was constructed and then co-transfectedwith HIV-1 based plasmid which containing the firefly luciferase reporter gene,into HEK293T cells to prepare the pseudotyped SARS-CoV-2 virus(ppSARS-2).We have successfully established the pseudotyped SARS-CoV-2 system for neutralization and entry inhibition assays.Huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model.Different levels of neutralizing antibodies were detected in convalescent serum samples of COVID-19 patients using ppSARS-2.The recombinant,soluble,angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppSARS-2 in Huh7.5 cells effectively.Furthermore,the neutralization results for ppSARS-2 were consistent with those of live SARS-CoV-2 and determined using the serum samples fromconvalescent patients.In conclusion,we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against SARS-CoV-2 and the entry process of the virus in a BSL-2 laboratory.