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