Synergistic effects of PA (S184N) and PB2 (E627K) mutations on the increased pathogenicity of H3N2 canine influenza virus infections in mice and dogs

Abstract

As companion animals, dogs are susceptible to various subtypes of influenza A virus (IAV), with the H3N2 and H3N8 subtypes of canine influenza virus (CIV) stably circulating among canines. Compared to the H3N8 CIV, the H3N2 CIV is more widely prevalent in canine populations and demonstrates increased adaptability to mammals, potentially facilitating cross-species transmission. Therefore, a comprehensive elucidation of the mechanisms underlying H3N2 CIV adaptation to mammals is imperative. In this study, we serially passaged the GD14-WT strain in murine lungs, successfully establishing a lethal H3N2 CIV infection model. From this model, we isolated the lethal strain GD14-MA and identified the key lethal mutations PA(S184N) and PB2(E627K). Moreover, the GD14-ma[PA(S184N)+PB2(E627K)] strain exhibited markedly enhanced pathogenicity in dogs. Viral titers in lung tissues from infected dogs and mice showed that GD14-ma[PA(S184N)+PB2(E627K)] does not increase its pathogenicity to mice and dogs by upregulating viral titers compared to the GD14-WT strain. Notably, sequence alignments across all H3N2 IAVs showed an increasing prevalence of the PA (S184N) and PB2 (E627K) mutations from avian to human hosts. Finally, single-cell RNA sequencing of infected mouse lung tissues showed that GD14-ma[PA(S184N)+PB2(E627K)] effectively evaded host antiviral responses, inducing a robust inflammatory reaction. Considering the recognized role of the PB2 (E627K) mutation in the mammalian adaptation of IAVs, our findings underscore the importance of ongoing surveillance for the PA (S184N) mutation in H3N2 IAVs.IMPORTANCESince the 21st century, zoonotic viruses have frequently crossed species barriers, posing significant global public health challenges. Dogs are susceptible to various influenza A viruses (IAVs), particularly the H3N2 canine influenza virus (CIV), which has stably circulated and evolved to enhance its adaptability to mammals, including an increased affinity for the human-like SAα2,6-Gal receptor, posing a potential public health threat. Here, we simulated H3N2 CIV adaptation in mice, revealed that the synergistic PA(S184N) and PB2(E627K) mutations augment H3N2 CIV pathogenicity in dogs and mice, and elucidated the underlying mechanisms at the single-cell level. Our study provides molecular evidence for adapting the H3N2 CIV to mammals and underscores the importance of vigilant monitoring of genetic variations in H3N2 CIV.

Keywords: H3N2 canine influenza virus; cross-species transmission; host adaptation; single-cell RNA sequencing.

Fig 1

Establishment of a lethal H3N2 CIV infection model in mice. (A and B) Body weight changes and survival rate of 4-week-old mice. (C and D) Body weight changes and survival rate of 5-week-old mice. (E and F) Body weight changes and survival rate of 6-week-old mice. (G and H) Body weight changes and survival rate of mice infected with plaque-purified viral strains. (Five mice per group; mice were considered to have succumbed if they lost more than 25% of their initial body weight). (I) Lung viral titers at 3 and 5 dpi. The dashed line signifies the lower limit of detection. (J) Histological assessment of lung sections from experimental mice at 3 and 5 dpi via hematoxylin-eosin (HE) staining and immunohistochemistry (IHC).

Fig 2

Synergistic lethal effects of PA (S184N) and PB2 (E627K) mutations in GD14-MA murine infections. (A and B) Body weight changes and survival rate of mice infected with single-segment replacement recombinant viruses. (C and D) Body weight changes and survival rate of mice infected with double-segment replacement recombinant viruses. (E and F) Body weight changes and survival rate of mice infected with double and triple-mutation recombinant viruses. (G and H) Body weight changes and survival rate of mice infected with GD14-ma[PA(S184N)] strains. (Five mice per group; mice were considered to have succumbed if they lost more than 25% of their initial body weight). (I) Prevalence of the PA (S184N) and PB2 (E627K) mutation across H3N2 subtypes of influenza A virus. (J) Polymerase activity, an empty plasmid vector devoid of the PA gene sequence was utilized as a negative control. (K) Lung viral titers at 3 and 5 dpi. The dashed line signifies the lower limit of detection. (L) Histological assessment of lung sections from experimental mice at 3 and 5 dpi via HE staining and IHC.

Fig 3

Enhanced pathogenicity of GD14-ma[PA(V100I)+PB2(E627K)] strain in dogs. (A) Clinical symptom scores in dogs. (B) Rectal body temperature measurements in dogs. (C) Viral titer in nasal swabs collected daily from 1 to 7 dpi. (D) Serum antibody titers in dogs. (E) Viral titers in the lungs, trachea, and turbinates at 3 dpi. (F) Gross appearance of the canine lungs. Black arrows delineate regions characteristic of pulmonary emphysema, whereas red arrows highlight areas indicative of hemorrhagic infiltration. (G) Histopathological examination of lung sections at 3 dpi using HE (the black arrow delineates the lesion site) staining and IHC.

Fig 4

Evasion of host antiviral defenses by the GD14-ma[PA(S184N)+PB2(E627K)] strain. (A) Schematic overview of the study design. A single mouse per group was euthanized at 3 dpi, and individual samples were subjected to scRNA-seq analysis. (B) t-SNE plot illustrating the formation of 11 major clusters from 21,589 suspended lung cells. (C) Cell type annotation and dot plot. (D) Total UMI counts of host genes associated with the antiviral response (GO: 0051607) across different cell clusters (x-axis). Dots represent cells from various clusters, colored according to the samples. (E, F) Normalized expression of ranked DEGs. Histograms display significantly upregulated DEGs (log2 fold change ≥0.5, P ≤ 0.05) enriched in the “defense response to virus” (GO: 0051607) for each cell type. Genes are ranked from top to bottom based on their average expression levels in GD14-WT-infected cells (E) or GD14-ma[PA(S184N)+PB2(E627K)] infected cells (F) compared to control cells within each cell type.

Fig 5

Elevated inflammatory responses and cytokine storms elicited by the GD14-ma[PA(S184N)+PB2(E627K)] strain in murine pulmonary infection. (A) UMI counts of host genes related to the “inflammatory response” (GO: 0006954) are shown across different cell clusters on the x-axis. Dots represent individual cells from various clusters, differentiated by color according to their sample groups. (B, C) Normalized expression levels of highly ranked DEGs. Histograms illustrate significantly upregulated DEGs (log2 fold change ≥0.5, P ≤ 0.05) that are enriched for the “inflammatory response” (GO: 0006954) in each cell type. The genes are ranked from top to bottom based on their average expression levels in GD14-WT-infected cells (B) or GD14-ma[PA(S184N)+PB2(E627K)]-infected cells (C) relative to control cells within the same cell type. (D) Predicted interaction map depicting the communication network mediated by CCL6 following challenge with GD14-ma[PA(S184N)+PB2(E627K)] strain.

Fig 6

Host cell infection patterns and viral transcript expression in murine lung tissue infected with CIV at single-cell resolution. CIV-susceptible cells were classified into highly infected (H, total UMI count ≥8 with expression of each viral segment), lowly infected (L, total UMI count ≥1), and undetected (U, UMI count = 0) based on CIV transcript expression counts. (A–C) T-SNE plots illustrate the expression of viral genes at the single-cell level. (D) The y-axis displays the percentages of cells susceptible to CIV infection, categorized as highly infected, lowly infected, and undetected cells. (E, F) Violin plots represent the distribution of gene expression levels for GD14-WT (E) and GD14-ma[PA(S184N)+PB2(E627K)] (F) within different cell populations, with the y-axis indicating the relative expression levels.