Purpose: The diversity of subtypes within Influenza A has recently expanded with the identification of H17N10 and H18N11 from bats. In order to further study the tropism and zoonotic potential of these viruses, we have produced lentiviral pseudotypes bearing H17 and N10 glycoptoteins. Influenza pseudotypes are powerful tools that pernit safe, sensitive serology and other cell-based assays to be performed, including at high-throughput. Different permutations of these pseudotypes permit specific measurement of anti-HA head, anti-HA stalk and anti-NA directed antibodies enabling new antivirals and mAbs to be screened.
Methods & Materials: H17N10 lentiviral pseudotypes were produced by co-transfection of 293T/17 cells with HIV Gag-Pol, H17, N10, protease (to cleave HA), and luciferase-carrying vector plasmids. HA neutralization assays were carried out with mAbs on 96 well plates using a Glomax luminometer. IC50 were calculated using GraphPad Prism. A novel NA pseudotype-based ELLA assay was used for NA inhibition determination.
Results: These H17N10 pseudotypes were shown to be efficiently neutralized by the broadly neutralizing HA stalk monoclonal antibodies CR9114 and FI6. We confirm that H17 can infect MDCKII cells and that it does not use sialic acid as its cellular receptor, as pseudotypes bearing H17 HA glycoprotein are released into the cell supernatant in the absence of neuraminidase. H17 pseudotypes are also unable to transduce cells that are permissive to non-chiropteran influenza A and B pseudotypes. We demonstrate that N10 can facilitate H5 and H7 influenza pseudotype release in the absence of another source of neuraminidase. Despite this, the N10 protein shows no activity in an enzyme-linked lectin assay.
Conclusion: This lentiviral pseudotype system will permit extensive new research on bat influenza tropism, therapeutics, restriction and seroepidemiology, without the constraints or safety issues with producing replication competent virus, to which the human population is naïve.
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