Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Abstract

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.

Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019

“…The atypical pathogenicity, high and prolonged viral excretory titers, and the swift dispersal observed for the low-pathogenicity H3N1 virus ( 19 , 35 ) affecting poultry in Belgium during 2019 merited an in depth investigation of its dispersal dynamics. For this purpose, spatially explicit phylogeographic reconstruction on the basis of AIV whole-genome sequence analysis was used to supplement and validate the available descriptive contact tracing data collected during and after the epidemic.…”

Infectivity and transmissibility of an avian H3N1 influenza virus in pigs

“…The full genome sequence was determined by Oxford Nanopore Technologies MinION sequencing (accession numbers OP765329, OP765352, OP765398, OP417305, OP765492, OP765503, OP765504, OP765508). Sequence analysis, performed in MEGA X (version 10.1.7), showed a deletion in the stalk of the NA and a mutation in the NA that is associated with plasminogen recruitment [ 21 , 22 ].…”

“…The lack of these mutations in the PB2 gene of chB19 is likely a factor in the poor replication of chB19 in pig tissues. Nasal mucosa 0 Before the onset of the H3N1 epizootic, two notable NA gene mutations occurred: (1) the loss of a glycosylation site at amino acid position 130, allowing the recruitment of plasminogen and facilitating a systemic replication in chickens [22], and (2) a deletion in NA stalk. The latter feature is common after transmission of AIVs from waterfowl to poultry [37].…”

“…Typically, low pathogenic AIVs ae dependent on trypsin-like proteases for HA cleavage and subsequent host cell entry, which limits their tropism to tissues in which these proteases are present. The NA of H3N1 is able to recruit plasminogen for HA cleavage, facilitating systemic spread of the virus [ 22 ]. This H3N1 outbreak occurred in an area with a dense swine population.…”

Aviaire influenza in vogelvlucht

“…Door de mutatie kan het neuraminidaseplasminogeen binden, dat na activatie de klieving van het HA medieert. Plasminogeen en plasminogeenactivatoren zijn alom vertegenwoordigd in het lichaam, ook in de hersenen, waardoor dit virus in staat is om zich systemisch te verspreiden (Schön et al, 2021). De vraag kan gesteld worden of de huidige opgelegde testen afdoende zijn om de pathogeniteit van alle AIstammen correct in te schatten.…”