H7N9 bearing a mutation in the nucleoprotein leads to increased pathology in chickens

Abstract

The zoonotic H7N9 avian influenza (AI) virus first emerged in 2013 as a low pathogenic (LPAI) strain, and has repeatedly caused human infection resulting in severe respiratory illness and a mortality of ~39% (>600 deaths) across five epidemic waves. This virus has circulated in poultry with little to no discernible clinical signs, making detection and control difficult. Contrary to published data, our group has observed a subset of specific pathogen free chickens infected with the H7N9 virus succumb to disease, showing clinical signs consistent with highly pathogenic AI (HPAI). Viral genome sequencing revealed two key mutations had occurred following infection in the haemagglutinin (HA 226 L>Q) and nucleoprotein (NP 373 A>T) proteins. We further investigated the impact of the NP mutation and demonstrated that only chickens bearing a single nucleotide polymorphism (SNP) in their IFITM1 gene were susceptible to the H7N9 virus. Susceptible chickens demonstrated a distinct loss of CD8⁺ T cells from the periphery as well as a dysregulation of IFNγ that was not observed for resistant chickens, suggesting a role for the NP mutation in altered T cell activation. Alternatively, it is possible that this mutation led to altered polymerase activity, as the mutation occurs in the NP 360-373 loop which has been previously show to be important in RNA binding. These data have broad ramifications for our understanding of the pathobiology of AI in chickens and humans and provide an excellent model for investigating the role of antiviral genes in a natural host species.

Keywords: H7N9; Mutation; T cell; influenza; interferon.

Figure 1

Unexpected deaths following H7N9 infection. Survival curve (A) and RT-qPCR detection of H7N9 systemic infection (B) following ONO inoculation of SPF chickens with A/Anhui/1/13 (H7N9). (A) Following inoculation of ten 16-week old SPF chickens with 10^7.7 EID₅₀ of A/Anhui/1/13 (H7N9), five chickens either died or were humanely killed between day 5 and 8 pi. (B) Swab and tissue samples taken upon necropsy from the five chickens that died/were humanely killed, were subjected to RT-qPCR analysis of the influenza M gene to assess the degree of systemic infection in the affected chickens.

Figure 2

H7N9 avian influenza viral localisation in tissues of 16 week-old chickens inoculated via the ONO route. (A) Kidney (Chicken 11), showing viral antigen in renal tubules (arrowheads). Viral infection is also associated in some regions with localised necrosis of renal interstitium and tubules (*). (B) Kidney (Chicken 11), higher magnification of the same tissue, showing viral antigen in the epithelium of the tubules. While some tubules appear healthy (arrows), others are distended with necrotic material (arrowheads). (C) Heart (Chicken 11), showing that viral antigen is mainly within capillary endothelium (arrowheads) and in one cardiomyocyte (arrow). (D) Heart (Chicken 11), higher magnification of the same tissue, showing detail of viral antigen in capillaries. Enucleated erythrocytes can be seen lined up within some of the capillaries. (E) Lung (Chicken 5), showing viral antigen within single cells, presumed to be macrophages, within the lung parenchyma. (F) Viral antigen is present in the yolk material (*) and inflammatory exudate within the airsac overlying the pancreas (bottom right of the image) and in the airsac membrane overlying the inflamed serosal surface of the duodenum (arrow) (Chicken 11). Immunohistochemistry for influenza nucleoprotein (red-brown pigment), counterstained with haematoxylin.

Figure 3

A mutation in the NP correlated with disease outcome. (A) The table shows a summary of the results of genome sequencing of virus isolated from H7N9 infected chickens. Key, known mutations, including presence absence of the MBCS associated with pathogenesis are shown. Changes are highlighted in red. (B) NGS quantification of two key mutations demonstrates the abundance (shown as percentage) of each mutation from the inoculum and a heart isolate from an infected chicken (chicken #9). (C) The sequence logo shows the abundance of different amino acids from available Genbank sequences for aa369-378 of the influenza NP protein (D) and predicted protein folding of the influenza NP protein showing amino acid 373 (T) (in red) on a solvent exposed side chain.

Figure 4

The 373T NP virus isolate causes severe disease and death but not the 373A NP. Survival curve (A), RT-qPCR (B) histopathology scores, qPCR (C) and live virus detection (D), of H7N9 systemic infection, following intravenous inoculation of SPF chickens with NP 373A and NP 373T bearing variants of A/Anhui/1/13 (H7N9). (A) Following inoculation of eight 16-week old SPF chickens with a 1:10 dilution of allantoic fluid containing live A/Anhui/1/13 (H7N9) bearing NP 373T, five chickens were humanely killed between day 2 and 3 pi. In contrast, following inoculation of eight 16-week old SPF chickens with a 1:10 dilution of allantoic fluid containing live A/Anhui/1/13 (H7N9) bearing NP 373A, all eight chickens survived until the predetermined endpoint (day 10 pi). (B) histopathology scores are presented for various tissues taken from susceptible chickens. Swab and tissue samples taken upon necropsy from the five chickens infected with the NP 373T bearing variant of A/Anhui/1/13 (H7N9), that were prematurely humanely killed, were subjected to RT-qPCR analysis of the influenza M gene (C) and live virus titration in MDCK cells (D) to assess the degree of systemic infection in the affected chickens.

Figure 5

Pathology and H7N9 antigen tropism in chickens inoculated with the NP 373T bearing variant of A/Anhui/1/13 (H7N9) by the intravenous route. (A) Heart (chicken 5) showing viral antigen capillary endothelium (arrowheads) and in a single myocardial fibre (arrow); tissue morphology is normal in this heart. (B) Lung (Chicken 8) showing viral antigen within single cells; tissue morphology is normal in this tissue. (C) Kidney (Chicken 4) showing acute interstitial and tubular necrosis, haemorrhage and histiocytic infiltration (*). (D) Kidney (Chicken 4; consecutive section to that shown in (C) showing viral antigen in tubular epithelium (arrowheads) and localised necrosis (*). Immunohistochemistry for influenza nucleoprotein (red-brown pigment), counterstained with haematoxylin (A, B, D); haematoxylin and eosin stain (C).

Figure 6

Severe disease is associated with severe CD8⁺ T cell depletion. Flow cytometry analysis of the proportions of (A) CD3⁺ T cells and (B) CD8⁺ cells comparing healthy uninfected control chickens to susceptible and resistant chickens. Cytokine expression analysis as determined by qPCR, in the lung and spleen of chickens infected with the (C) NP 373T bearing variant of A/Anhui/1/13 (H7N9) compared chickens infected with the (D) NP 373A bearing variant of A/Anhui/1/13 (H7N9). Error bars represent SEM. *p<0.05, **p<0.01.