Development of LPAI and HPAI H7 avian influenza pseudotypes for serological applications utilising a combination of protease cotransfection and site-directed mutagenesis

Molesti, Eleonora and Cattoli, Giovanni and Ferrara, Francesca and Böttcher-Friebertshäuser, Eva and Terragino, Calogero and Temperton, Nigel J. (2013) Development of LPAI and HPAI H7 avian influenza pseudotypes for serological applications utilising a combination of protease cotransfection and site-directed mutagenesis. In: Society for General Microbiology Spring Meeting, 25-28 Mar 2013, Manchester. (Unpublished) (doi:MA09/07) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

Abstract

HPAI and LPAI H5 and H7 viruses have been disastrous for the economies of affected countries reliant on poultry production. In a situation similar to H5, H7 strains show adaptation to humans and therefore pose a serious public health concern. Applying knowledge acquired from study of H5 virus evolution and spread to the development of sensitive serological methods will improve our ability to understand and respond to the emergence of other HPAI and LPAI viruses with pandemic potential. We describe the production of pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian influenza for use as tools in pseudotype-based (pp-NT) neutralisation assays. A crucial feature of H7 pseudotype production is efficient intracellular cleavage of haemagglutinin. We show that the LPAI strain A/chicken/Italy/1082/1999 possesses a monobasic cleavage site and requires TMPRSS2 to effect cleavage of the HA. The HPAI strain A/ Pakistan/34668/95 possesses a sub-optimal furin cleavage site resulting in low yields. In order to circumvent this we have used site-directed mutagenesis to replace the polybasic cleavage site with a monobasic site that can subsequently be cleaved (during production) via the cotransfection of the TMPRSS2 protease. Sensitive pp-NT assays were then carried out on post-vaccination sera using these new surrogate viruses.

Item Type:	Conference or workshop item (Poster)
Subjects:	Q Science > QR Microbiology > QR355 Virology
Divisions:	Faculties > Sciences > Medway School of Pharmacy
Depositing User:	Nigel Temperton
Date Deposited:	22 Apr 2013 14:09 UTC
Last Modified:	09 Jun 2014 15:58 UTC
Resource URI:	https://kar.kent.ac.uk/id/eprint/33654 (The current URI for this page, for reference purposes)