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

By Eleonora Molesti, Giovanni Cattoli, Francesca Ferrara, Eva Böttcher-Friebertshäuser, Calogero Terragino and Nigel J. Temperton


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

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