Dynamic adaptation of A(H3N2) virus in mice
To obtain an A(H3N2)-MA strain, we conducted successive lung-to-lung passages of the A(H3N2)-WT strain in C57BL/6J mice. The A(H3N2) viruses identified in 17 passages were designated as MA1–MA17, respectively (Fig. 1A). According to the changes in body weight, survival state, lung index (a ratio of the weight of the lung to the body weight of the mouse), and lung viral RNA loads of the infected mice, MA1, MA5, MA8, MA13, and MA17 were selected as key passages in the following analysis.
Adaptation of human seasonal H3N2 influenza virus in mice. A Schematic diagram of the lung-to-lung passage experiment of A(H3N2) virus and pathogenicity test of the A(H3N2)-MA and A(H3N2)-WT strains in the mouse model. B Change in body weight, (C) survival rate, and (D) lung index of mice were monitored during key passages (MA1, MA5, MA8, MA13, and MA17). The dotted line in panel B represents 75% of the initial body weight. E Virus loads in the lungs of infected mice during key passages were detected by qRT-PCR. The dashed horizontal line indicates the lowest limit of detection. Each group includes three mice. Grey asterisks represent statistical significance between the infected group and the control group (intranasally inoculated with an equal volume of sterile PBS), while blue asterisks indicate significant differences between the MA1 group and other groups. Statistical significance among groups was analyzed using Kruskal–Wallis test with Dunn’s multiple comparisons (D, E). Error bars show the mean ± SD. *, P < 0.05; **, P < 0.01
In the MA1, no body weight loss was observed in mice infected with the A(H3N2)-WT strain (Fig. 1B), and all mice survived during the observation period (Fig. 1C). The lung indexes of the infected mice showed no noticeable changes compared to the controls (Fig. 1D), and virus was detected in one of the three mice at 5 dpi. In the MA5, the infected mice have a decrease of approximately 5% in body weight at 7 dpi and later the body weight returned to a normal state. No mice died, but the lung indexes and lung virus RNA loads of the infected mice were higher than those of the control group (Fig. 1B–E).
As the number of passages increased, the body weight losses of the infected mice increased compared with their initial body weights before virus infection. Specifically, from 5 to 7 dpi, the rates of body weight reduction were greater than 22% for MA8, 25% for MA13, and 25% for MA17 compared to their initial body weights (Fig. 1B). In addition, in the MA8 passage, the survival rate of the infected mice dropped to 33%, while all mice in the MA13 and MA17 passages died within 7 and 5 dpi, respectively (Fig. 1C). Additionally, the lung indexes of the infected mice gradually increased as passage increased. The lung indexes for mice from the MA13 and MA17 passages were significantly higher than that of the control group, respectively (Fig. 1D). Meanwhile, the lung virus RNA loads of mice from the MA5, MA8 and MA13 passages progressively increased. The mean virus RNA loads of MA17 was comparable with that of the MA13 passage, which were both significantly higher than that of MA1 (Fig. 1E). These results suggest that as the passage number increased, the A(H3N2) virus from the infected mice showed gradual enhancement in their virulence, replication ability, and adaptation to mice.
Adaptation of A(H3N2) in mice involves dynamic mutations of the virus
To reveal the molecular basis correlated to the adaptation of the A(H3N2) virus in mice, we analyzed AA mutations that occurred in the viruses from five key passages (MA1, MA5, MA8, MA13, and MA17). This analysis was based on the genomic data generated using NGS method. We identified fourteen mutations that are unique in the A(H3N2)-MA virus rather than the A(H3N2)-WT strain (Table 1). These AA mutations include twelve nonsynonymous mutations (PB2-S590R, PB1-I682V, PA-K615E, PA-X-F246S, HA-N91T, HA-N122D, HA-K207E, HA-I242T, HA-N246K, HA-M478I, NP-G384R, and M1-D232N) in PB2, PB1, PA, PA-X, HA, NP, and M1 genes, and two synonymous mutations (PA-G(GGG)462G(GGA) and PA-L(CTT)246L(CTC)) in PA gene.
Among these, four mutations are prone to occur during the mice infection. The HA-N246K (H3 numbering throughout), HA-M478I, NP-G384R, and M1-D232N mutations occurred in the MA1 passage, with a higher proportion of greater than 90%. In addition, six of the fourteen mutations (i.e., PB2-S590R, PB1-I682V, PA-K615E, PA-X-F246S,PA-G(GGG)462G(GGA), and PA-L246L) have a gradual accumulation progress during the passaging. The PB2-S590R, PA-K615E, and PA-G(GGG)462G(GGA) mutations occurred in the MA1 passage, with proportions of 1.47%, 1.27%, and 7.56%, respectively, and their proportions reached 97.73%, 98.57%, and 98.59% in the MA17 passage, respectively. The PB1-I682V, PA-X-F246S, and PA-L(CTT)246L(CTC) mutations were first found in the MA5, MA8, and MA8, respectively. Furthermore, three mutations on the HA genes occurred at the late stage of the virus adaptation, and the HA-N91T, HA-K207E, and HA-I242T mutations were first observed in the MA13 passage, with proportions of 11.6%, 11.16%, and 11.18%, respectively; and these mutations increased to approximately 70% in the MA17 passage. In addition, the HA-N122D had already occurred in the MA5 passage with a proportion of 97.75% but was not found in the MA1 passage.
Moreover, eight mutations, PB2-S590R, PA-K615E, HA-N122D, HA-N246K, HA-M478I, NP-G384R, M1-D232N, and PA-G(GGG)462G(GGA) with the proportion greater than 95% were kept in the MA17 passage. In addition, the other mutations took an occupation of greater than 50% in the MA17 passage.
Mouse adapted mutation enhances the polymerase activity of the RNP complex
To investigate the impact of mouse-adapted mutations in the PB2, PB1, PA, and NP genes on polymerase activity, we analyzed the activity of the RNP complex from these gene combinations. As shown in Fig. 2, the relative polymerase activity of the RNP complex containing PB2-S590R, PB1-I682V, PA-K615E, or NP-G384R ranged from 120 to 136%, which is higher than that of the A(H3N2)-WT, set at 100%. Specifically, the polymerase activity of the combination with PA-K615E and NP-G384R, as well as the combination with PB2-S590R and PA-K615E, was greater than that of A(H3N2)-WT. The activity levels of the combination of PB2-S590R, PA-K615E, and NP-G384R and the combination of PB2-S590R, PB1-I682V, and PA-K615E were comparable and significantly higher than A(H3N2)-WT (P < 0.05). Ultimately, the polymerase activity of the combination with PB2-S590R, PB1-I682V, PA-K615E, and NP-G384R was also significantly higher than A(H3N2)-WT (P < 0.05). These results indicated that the mouse-adapted mutations in the PB2, PB1, PA, and NP genes enhanced the polymerase activity, which may affect the replication and virulence of A(H3N2) virus in mice.
Polymerase activity of ribonucleoprotein (RNP) complexes of the A(H3N2)-WT and A(H3N2)-MA strains. A The polymerase activities of the RNP complexes for the WT and MA strains were detected using a dual-luciferase reporter system with PB2, PB1, PA, and NP expression vectors in HEK-293T cells. The blue and red rectangles represent the gene from A(H3N2)-WT and A(H3N2)-MA, respectively. Values represent the mean ± SD from three independent experiments and are standardized to those of A(H3N2)-WT measured in HEK-293T cells. Blue asterisks indicate statistical significance between the wild-type group and the other groups. Statistical significance between groups was analyzed by the Kruskal–Wallis test with Dunn’s multiple comparisons. *, P < 0.05. B NP protein expression was detected in the HEK-293T cell lysis supernatant by Western blotting
Pathogenic and replication characteristics of A(H3N2)-MA and A(H3N2)-WT strains in mice
The mouse-adapted A(H3N2) strain, A/Kansas/14/2017/H3N2-MA, was generated through three rounds of plaque purification of the viruses from lung homogenate of the MA17 passage in MDCK cells. We first investigated the pathogenic characteristics of the A(H3N2)-MA and A(H3N2)-WT strains based on the MLD50 (Fig. 1A). The mice were infected with the A(H3N2)-WT strain of 108 EID50 and the A(H3N2)-MA strain of different infection doses (from 103 to 106 EID50, to tenfold dilution), respectively. The results showed that mice infected with A(H3N2)-MA of 103 EID50 experienced a 10% decrease in body weight at 7 dpi, and later began to increase. The body weights of mice infected with 104 EID50 of A(H3N2)-MA decreased by approximately 20% at 8 dpi, and two of five mice died at 9 dpi (Fig. 3A and B). Most mice infected with 105 EID50 and 106 EID50 of A(H3N2)-MA lost body weights of greater than 25% at 7 dpi, and all mice succumbed at 8 and 7 dpi, respectively. In contrast, mice infected with the maximum titer of 108 EID50 of A(H3N2)-WT showed no significant body weight loss, and all five mice survived (Fig. 3A and B). Therefore, the MLD50 value for the A(H3N2)-MA strain is 104.167 EID50/50 μL, and for the A(H3N2)-WT is greater than 108.0 EID50/50 μL, indicating that the virulence of A(H3N2)-MA is higher than A(H3N2)-WT in the mouse model.
Pathogenicity and replicability of the A(H3N2)-WT and A(H3N2)-MA strains in mice. Five mice of each group were intranasally infected with tenfold serial dilutions containing 106 to 103 EID50 of A(H3N2)-MA or with a dose of 108 EID50 of A(H3N2)-WT, respectively. The control group was intranasally inoculated with an equal volume of sterile PBS. A Changes in body weight and (B) survival rates of mice were monitored for 14 dpi. In addition, two groups of 20 mice were intranasally inoculated with 104.5 EID50/50 μL A(H3N2)-MA or A(H3N2)-WT, respectively, and (C) body weight changes and (D) survival rate of mice were measured daily. Five mice per group were euthanized at 3, 5, and 7 dpi, respectively. Virus RNA loads in the lung (E) and nasal turbinate (F) of the mice were determined by qRT-PCR. The dashed horizontal lines indicate the lowest limit of detection (E, F). Statistical significance between groups was analyzed using Kruskal–Wallis test with Dunn’s multiple comparisons (E, F). The grey asterisks represent statistical significance between the A(H3N2)-MA and the control group. The blue asterisks represent statistical significance between the A(H3N2)-MA and A(H3N2)-WT groups. Error bars indicate mean ± SD. **, P < 0.01
Furthermore, we compared the pathogenicity of A(H3N2)-MA and A(H3N2)-WT in the mouse model infected with the same dose of 104.5 EID50/50 μL. In the A(H3N2)-MA group, the body weights gradually decreased to approximate 77% of initial weight at 8 dpi, and two of five mice died at 8 dpi, with a survival rate of 60% (Fig. 3C and D). Conversely, mice infected with A(H3N2)-WT exhibited no body weight loss compared to the control group, and all mice survived (Fig. 3C and D). Furthermore, the mean values of lung viral RNA loads of the mice infected with A(H3N2)-MA were 109.82, 1010.84, and 109.69 copies/mL at 3, 5, and 7 dpi, respectively (Fig. 3E), meanwhile virus loads in the nasal turbinate were 108.15, 108.91, and 108.17 copies/mL (Fig. 3F). In contrast, no viral RNA were detected in the lung and nasal turbinate of mice in the A(H3N2)-WT group at 3, 5, and 7 dpi. These findings indicated that the pathogenicity and replication capacity of the A(H3N2)-MA strain were significantly greater than those of the A(H3N2)-WT strain.
Tissue tropism of A(H3N2)-MA and A(H3N2)-WT in the mouse model
To evaluate tissue tropism of the A(H3N2)-MA and -WT strains, we also measured the virus RNA loads in seven tissues (including the heart, liver, spleen, kidney, intestine, stomach, and brain) from the infected mice with 104.5 EID50 at 3, 5, and 7 dpi. The results exhibited that no viral RNA was detected in these seven tissues, regardless of the A(H3N2)-MA or A(H3N2)-WT strains (Fig. S1). These findings suggested that the tissue tropism of A(H3N2)-MA has not changed compared to the WT strain, although the replication ability of the MA strain significantly increased in the respiratory system.
Lung pathology and cytokine changes in mice infected with A(H3N2)-MA and A(H3N2)-WT
Next, we compared the pathological and inflammatory factor changes in the lungs of the mice infected with A(H3N2)-MA and A(H3N2)-WT viruses. At 7 dpi, lung tissue infected with A(H3N2)-MA showed more severe pathological damage than those with A(H3N2)-WT. In the A(H3N2)-MA group, greater infiltration of inflammatory cells into the alveoli, blood vessels, and bronchioles, as well as increased shedding of bronchiolar epithelial cells were observed (Fig. 4A). Consistently, the histopathological score for the A(H3N2)-MA group was significantly elevated compared with the A(H3N2)-WT group. But no significant difference was observed between the A(H3N2)-WT and the control groups (Fig. 4B).
Lung histopathology and inflammatory factors in mice infected with A(H3N2)-MA and A(H3N2)-WT. Mice were intranasally infected with 104.5 EID50/50 μL A(H3N2)-MA or A(H3N2)-WT, respectively. The control mice were intranasally inoculated with an equal volume of sterile PBS. A Histopathological changes in the lungs of mice were analyzed at 7 dpi. The scale bar represents 100 μm, and the magnification is 100x. B Histopathological scores for the lungs at 7 dpi. The levels of cytokines and chemokines (C) IL-6, (D) TNF, (E) MCP-1, and (F) IFN-γ in the lungs of mice were measured at 3, 5, and 7 dpi, respectively. Five mice were in each group. Statistical significance between groups was analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons (B, C, D, E, F). The grey asterisks represent statistical significance between the A(H3N2)-MA and the control group. The blue asterisks represent statistical significance between the A(H3N2)-MA and A(H3N2)-WT groups. Error bars represent mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001
We then assessed the levels of cytokines and chemokines in the mouse lungs from the A(H3N2)-MA, A(H3N2)-WT, and control groups. Pro-inflammatory factors including IL-6 (Fig. 4C), TNF (Fig. 4D), and MCP-1 (Fig. 4E) were significantly higher in the A(H3N2)-MA group than in the A(H3N2)-WT group at 3, 5, and 7 dpi. Additionally, the level of the pro-inflammatory factor, IFN-γ, was significantly higher in the A(H3N2)-MA group compared with the A(H3N2)-WT group at 5 dpi and 7 dpi (Fig. 4F). These results suggested that the infection of A(H3N2)-MA induced more severe lung pathological damage and inflammatory response than the A(H3N2)-WT.