Fig 1.
Intravenous infection of chickens with Belgian H3N1 isolates: Clinical course, HI-titers, and virus antigen detection.
Chicken were infected intravenously (i.v.) with either (A) CK/DE/1940/19 or (B) DK/BE/1358/19 H3N1 isolate. The clinical status was monitored over a period of ten days post inoculation (dpi). Clinical status is recorded as healthy (0), birds showing slight (1) or severe disturbances of the general condition (2). In both groups, one animal showed neurological signs including torticollis and was euthanized, as indicated with asterisk. Subsequently they were recorded as dead (3). The IVPI is the mean score per bird per observation over the 10-day period (1 observation per bird per day). In addition, virus shedding was measured and presented here as infectious virus equivalents (VE) per ml, calculated on basis of cq values from combined oro-pharyngeal swabs and correlated to a standard curve of stock virus with known infectivity titer. VE below 1 are indicated with (+) and VE of 0 are indicated with (-). Finally, antibody HI-titers of sera were determined three weeks after infection. Viral matrix protein detection in the (C) brain stem and (D) cerebral cortex after i.v. infection with CK/BE/1940/19. (E) No antigen detection following i.v. TK/BE/1358/19 infection, despite significant inflammatory infiltration. Influenza virus matrix protein was detected by immunohistochemistry, using the ABC method, AEC as chromogen (red) and counterstained by haematoxylin (blue). Bar = 50 μm (C, D) and 100 μm (E).
Fig 2.
Intracerebral inoculation of chickens with H3N1 AIV: Clinical course and virus genome in organs.
Day-old SPF chicks were inoculated intracerebrally either with (A) CK/BE/1940/19 (H3N1) or with (B) DK/DE/2555/06 (H3N1) virus isolates. The health status was scored daily over the period of eight days post inoculation (dpi), with (0) as healthy, (1) as diseased and (2) as dead. The ICPI is calculated as the mean score per bird per observation over the 8-day period (1 observation per bird per day). Organ samples were taken on day of death or 8 dpi and analyzed for virus genome load, presented here as infectious virus equivalents (VE)/ml, calculated by correlation of cq values to a standard curve of the respective virus stock with known titer.
Fig 3.
Tissue tropism of DK/DE/2555/06 and CK/BE/1940/19 following ICPI and inoculation of embryonated chicken eggs.
(A) Representative images of IAV matrix protein (M1) detected in brain (a-c), lung (d-f), kidney (g-i) and intestine (j-l), following intracerebral inoculation of day-old chicks; time points indicate days post inoculation (dpi). ABC method, AEC chromogen (red), counterstain haematoxylin (blue). Bar = 50 μm. (B) Summary of semi-quantitative antigen detection in parenchymal cells of indicated organs. Dots represent individual animal tissue scores: 0 = no antigen, 1 = focal to oligofocal, 2 = multifocal, 3 = coalescing/diffuse. Bar indicates median, GIT = gastrointestinal tract. Details given in S1 Fig. (C) Immunohistochemical detection of IAV matrix protein (M1) in the CAM following allantoic inoculation of 10-day old embryonated SPF chicken eggs with either (a) DK/DE/2555/06 or (b) Ck/BE/1940/19 isolate. Indicated chorionic (pink asterisk) and allantoic epithelium (green asterisk). ABC method, AEC chromogen (red), counterstain haematoxylin (blue). Bar = 50 μm.
Fig 4.
No furin cleavage of CK/BE/1940/19 (H3N1) HACS determined by luciferase reporter assay.
Comparative cleavability of the hemagglutinin proteolytic cleavage site by furin or other intra-Golgi proteases, was determined by luciferase reporter assay in QM9 (avian) and A549 (human) cells. No signal was detected for the negative control, represented by a sequence with no HACS motif. For the HPAIV-derived polybasic HACS control a robust signal, more abundant in A549 cells, was generated. The CK/BE/1940/19 derived HACS, as well as a modified one with exchange of lysine at P1 against the more common arginine, did not induce signals indicating proteolytic processing.
Fig 5.
Sequence comparisons and phylogenetic analysis of the CK/BE/1940/19 neuraminidase (N1).
(A) Amino acid alignment of positions described as responsible for neuraminidase mediated plasminogen binding. The loss of the Asn at position 130 and therefore the loss of the N-glycosylation site (blue isolate name), as described for WSN, has also been found in the CK/BE/1940/19 isolate but not in the comparative DK/DE/2555/06 isolate (red dots). First Belgian H3N1 isolate from January still has intact N-glycosylation site (green dot). Database research (www.fludb.de) including all full length N1-sequences without duplicates reveals only 24 of 11417 sequences to have no asparagine at this position. These 24 isolates (listed in detail in S2 Table) are shown in the (B) phylogenetic tree (calculated as a maximum likelihood tree using RAxML with a bootstrap value of 1000 cycles) together with the available N1-sequences from the Belgian H3N1 outbreak (n = 9) (both blue, except the Belgian January isolate) from 2019 and other representative N1 scaffold sequences (black).
Fig 6.
Trypsin-independent spread and HA0 cleavage of CK/BE/1940/19 is blocked by 6-aminohexanoic acid.
(A) Immunofluorescent plaque assays on LMH cells were conducted with either DK/DE/2555/06 (H3N1) or CK/BE/1940/19 (H3N1) isolate. Cells were formalin fixated following incubation for 24 or 72 hours post inoculation (hpi). Cytopathogenic effect (CPE) was only observed for the CK/BE/1940/19 isolate by translucent light microscopy (left). Immunostaining confirmed IAV antigen is colocalized with visible CPE for the CK/BE/1940/10. Time-dependent increase of plaque size indicates ongoing virus spread. Only single individual cells stained positive with DK/DE/2555/06 and no increase in number of infected cells was detectable. (B) Trypsin-independent replication of CK/BE/1940/19 can be blocked by 6-aminohexanoic acid (AHA) supplementation in immunofluorescence plaque assays. While the LPAIV reference virus DK/DE/2555/06 only infect single cells without trypsin supplementation, the CK/BE/1940/19 H3N1 efficiently spread without trypsin and/or argatroban supplementation, a thrombin specific inhibitor. In contrast when cultivated with AHA, a plasmin specific inhibitor, CK/BE/1940/19 becomes restricted to single cell infection. (C) In vitro proteolytic cleavage of HA0 without trypsin-supplementation was prevented by AHA as shown by western blot. (left) Ponceau S stain proteins unspecific, confirming comparable protein loads of cell lysate at the membrane. (right) Chemiluminescent detection following incubation with H3 specific antibody (binding within HA1). There is a clear band at size of 70 kDa for the cell lysates of CK/BE/1940/19 and DK/DE/2555/06 inoculated cells, which representing the uncleaved HA0 protein. For CK/BE/1940/19, there is an additional band right below 55 kDa, representing the HA1, indicating proteolytic cleavage of HA0. This band do not appear when cultivated with AHA supplementation. In contrast, the allantoic fluid stock of CK/BE/1940/19 only, exhibits a strong HA1 specific signal.