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

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.