Cloning and Characterization of a Low Pathogenic H7N7 Avian Influenza Virus with an Artificial Multibasic Cleavage Site

In 2013 the LPAIV A/turkey/Germany/AR534/13 (H7N7) (wt-H7N7) was detected in commercial poultry flocks in German federal states North-Rhine Westphalia and Lower Saxony. As in many countries the outbreak was eradicated by culling of all infected poultry. It is known that HPAIV can arise in poultry from LPAIV. Therefore, the aim of this study was to assess the risk of conversion of this isolate to a highly pathogenic phenotype. A multibasic CS within the HA is regarded as prime determinant for pathogenicity. Thus, a recombinant LP isolate (LP-N7N7) was generated by reverse genetics and the respective HP virus was produced by the introduction of an artificial multibasic CS within the HA (HP-H7N7). While HA cleavage and plaque formation of HP-H7N7 was independent from the addition of an exogenous protease in vitro, efficiency of HA cleavage and plaque formation of LP-H7N7 increased in the presence of trypsin as for wt-H7N7. Replication kinetics of LP-H7N7 as well as HP-H7N7 were comparable to wt-H7N7. For assessment of virulence, chickens were infected with LP-H7N7 of HP-H7N7. An oculo-nasal infection of chickens with LP-H7N7 caused temporarily mild clinical signs, whereas the infection with HP-H7N7 resulted in death of all infected animals within three days.Therefore, the LPAIV A/turkey/Germany/AR534/13 (H7N7) contains cryptic virulence determinants within the genome, allowing that the replacement of the monobasic by a multibasic HA CS alone is sufficient for transformation of this LPAIV to a highly pathogenic phenotype.