Synergy between HA cleavage site sequence and NA-mediated plasminogen recruitment as a virulence mechanism for low-pathogenic avian influenza

An outbreak of H3N1 low-pathogenic avian influenza virus (LPAIV) in Belgium in 2019 caused unexpected levels of mortality and morbidity in poultry. These viruses possess an NA polymorphism associated with plasminogen (PLG) binding, as well as an atypical sequence around the HA cleavage site; accordingly, HA cleavage mediated by NA-driven PLG recruitment has been proposed to underlie their systemic spread and pathogenicity. To test this, we established a reverse genetics system for A/chicken/Belgium/460/2019 and created single mutations in HA (K345R) and NA (S122N) that restored the viruses to normal consensus, as well as an HA/NA double mutant. Confirming previous work, trypsin-independent spread and HA cleavage of wild-type Ck/Belgium were observed in the presence of fetal bovine serum containing PLG <i>in vitro</i>. Dose-dependent HA cleavage and trypsin-independent spread were also observed in the presence of purified chicken PLG. Compared to the wild-type virus, both HA cleavage and virus spread <i>in vitro</i> were reduced by the HA K345R mutation and further blocked by the NA mutation S122N. PLG-mediated HA cleavage was seen in a variety of avian cell lines and chicken organoids, excluding cell type-dependent effects. Furthermore, <i>in ovo</i> tests showed that mutant viruses unable to recruit PLG were less able to replicate systemically in chicken embryos. Bioinformatics analyses revealed other viruses that could potentially recruit PLG, including two independent outbreaks of H6N1 viruses, one of which we confirmed PLG-driven spread <i>in vitro</i>. We conclude that PLG recruitment by NA is a general virulence mechanism of N1 LPAIVs.IMPORTANCEAvian influenza viruses are divided into high or low pathogenicity based on the sequence of their hemagglutinin (HA) and their lethality in chickens. The majority of AIV strains circulating in the wild are of low pathogenicity both in waterfowl and when they spill over into domestic poultry. However, some low-pathogenicity strains can cause serious disease in poultry. A severe 2019 outbreak of an H3N1 strain has been suggested to result from the viral neuraminidase (NA) recruiting cellular plasminogen to proteolytically activate HA. Here, we confirmed that the sequence of the NA at position 122 is the primary determinant of plasminogen-driven HA cleavage, but that the unusual sequence at the HA cleavage also contributes to pathogenicity. Furthermore, we show that this N1 NA sequence motif can be used to identify other unexpectedly virulent AIV strains. This work, therefore, adds to our ability to risk assess AIV strains from sequence-based surveillance.