ying Xie

and 11 more

Plasmid-based reverse genetics has transformed influenza virus research by enabling the production of recombinant influenza viruses from cloned cDNA copies of viral genome segments. Reverse genetic production of influenza viruses requires cellular expression of influenza proteins polymerase basic 1, polymerase basic 2, polymerase acidic and nucleoprotein which collectively allow for transcription of viral mRNA and synthesis of new negative-sense genomic RNA, thus enabling synthesis of all components needed to assemble infectious virus from transfected cell lines. Given the importance of these proteins in the generation of influenza viruses via reverse genetics, we sought to explore how transgenic expression of mammalian-adapted PB1, PB2, PA, or NP in the 293T packaging cell line may impact the recovery of recombinant influenza viruses. We constructed four transgenic 293T cell lines expressing PB1, PB2, PA or NP derived from the mouse-adapted 2009 pandemic influenza A virus, UI182. Transgenic expression of UI182 PB2 in 293T cells enhanced recovery of replication-competent avian influenza viruses generated by reverse genetics relative to levels achieved in unmodified 293T cells. Virus recovered from PB2-expressing 293T cells replicated with kinetics that were indistinguishable from viruses recovered from unmodified 293T cells. Provision of UI182 PB2 protein via transgenic expression in 293T cells resulted in enhanced viral polymerase activity as measured by a minigenome assay, which may account for the improved efficiency of viral packaging relative to unmodified 293T cells. Transgenic expression of mammalian-adapted PB2 in 293T cells may serve as an important tool for enhancing influenza virus recovery in reverse genetic systems.

Zengguo Cao

and 17 more

Ebolavirus (EBOV) is responsible for several EBOV disease (EVD) outbreaks in Africa, with a fatality rate of up to 90%. During 2014-2016, An epidemic of EVD spread throughout Sierra Leone, Guinea and Liberia, and killed over 11,000 people. EBOV began to circulate again in the Democratic Republic of Congo in 2018. Due to the need for a BSL-4 facility to manipulate this virus, the development and improvement of specific therapeutics has been hindered. As a result, it is imperative to perform reliable research on EBOV under lowered BSL restrictions. In this study, we developed a safe neutralization assay based on pseudotyped EBOV, which incorporates the glycoprotein of the 2014 EBOV epidemic strain into a lentivirus vector. Our results demonstrated that the tropism of pseudotyped EBOV was similar to that of authentic EBOV, but with only one infection cycle. And neutralizing activity of both authentic EBOV and pseudotyped EBOV were compared in neutralization assay using three different samples of antibody-based reagents against EBOV, similar results were obtained. In addition, an indirect ELISA was performed to show the relationship between IgG and neutralizing antibody against EBOV detected by our pseudotyped EBOV-based neutralization assay. As expected, the neutralizing antibody titers varied with the IgG titers detected by indirect ELISA, and a correlation between the results of the two assays was identified. By comparison with two different assays, the reliability of the results detected by the pseudotyped EBOV-based neutralization assay was confirmed. Collectively, in the absence of BSL-4 restrictions, pseudotyped EBOV production and neutralizing activity evaluation can be performed safely and in a manner that is neither labor- nor time-consuming, providing a simple and safe method for EBOV-neutralizing antibody detection and the assessment of immunogenicity of EBOV vaccines. All these remarkable advantages of the newly established assay highlight its potential to further application in assessment of immunogenicity of EBOV vaccine candidates.