Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.

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.

A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.

Comparison of the Immunogenicities of HIV-1 Mutants Based on Structural Modification of env

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

White spot syndrome virus envelope protein VP124 involved in the virus infection

White spot syndrome virus （WSSV） is one of the major shrimp pathogens causing large economic losses to shrimp farming. In an attempt to identify the envelope proteins involved in the virus infection, purified WSSV virions were mixed with three antisera against WSSV envelope proteins （VP39, VP124 and VP187 ）, individually. And then they were injected intramuscularly into crayfish （Procambarus clarkii） to conduct in vivo neutralization assays. The results showed that for groups injected with virions only and groups injected with the mixture of virions and antiserum against VP124, the crayfish mortalities were 100% and 60% on the 8th day postinfection, individually. The virus infection could be delayed or neutralized by antibody against the envelope protein VP124. Quantitative PCR was used to further investigate the influence of three antisera described above on the virus infection. The results showed that the antiserum against VP124 could restrain the propagation of WSSV in crayfish. All of the results suggested that the viral envelope protein VP124 played a role in WSSV infection.

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.