Multiple amino acid substitutions involved in the adaption of three avian-origin H7N9 influenza viruses in mice

Abstract

Background: Avian influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. The number of reported H7N9 virus human cases confirmed by laboratory has surpassed that of H5N1 virus. However, the mechanism for how H7N9 influenza virus overcomes host range barrier has not been clearly understood.

Methods: To generate mouse-adapted H7N9 influenza viruses, we passaged three avian-origin H7N9 viruses in mice by lung-to-lung passages independently. Then, the characteristics between the parental and mouse-adapted H7N9 viruses was compared in the following aspects, including virulence in mice, tropism of different tissues, replication in MDCK cells and molecular mutations.

Results: After ten passages in mice, MLD₅₀ of the H7N9 viruses reduced >750-3,160,000 folds, and virus titers in MDCK cells increased 10-200 folds at 48 hours post-inoculation. Moreover, the mouse-adapted H7N9 viruses showed more expanded tissue tropism and more serious lung pathological lesions in mice. Further analysis of the amino acids changes revealed 10 amino acid substitutions located in PB2 (E627K), PB1 (W215R and D638G), PA (T97I), HA (H3 numbering: R220G, L226S, G279R and G493R) and NA (P3Q and R134I) proteins. Moreover, PB2 E627K substitution was shared by the three mouse-adapted viruses (two viruses belong to YRD lineage and one virus belongs to PRD lineage), and PA T97A substitution was shared by two mouse-adapted viruses (belong to YRD lineage).

Conclusions: Our result indicated that the virulence in mice and virus titer in MDCK cells of H7N9 viruses significantly increased after adapted in mouse model. PB2 E627K and PA T97A substitutions are vital in mouse adaption and should be monitored during epidemiological study of H7N9 virus.

Keywords: H7N9 influenza virus; adaption; amino acid substitution; mouse; virulence.

Fig. 1.

Pathogenicity of the parental and mouse-adapted H7N9 viruses in mice. Groups of 5 6-week-old female BALB/C mice were inoculated i.n. with 10⁶ EID₅₀ (50 μl) of H7N9-53, H7N9-53 MA, H7N9-MCX, H7N9-MCX MA, H7N9-ZSM, H7N9-ZSM MA and PBS, respectively. Body weight changes and survival rates were monitored daily for 14 days. a Weight changes were recorded daily and represented by means (± standard deviation). b Mortality was determined by percentage of the surviving mice.

Fig. 2.

Titers of virus in different tissues of the parental and mouse-adapted H7N9 viruses in mice. Groups of 6 6-week-old female BALB/C mice were inoculated i.n. with 10⁶ EID₅₀ (50 μl) of H7N9-53, H7N9-53 MA, H7N9-MCX, H7N9-MCX MA, H7N9-ZSM, H7N9-ZSM MA and PBS, respectively. 3 dpi, 3 mice were euthanized to collect hearts, liver, spleens, lungs, kidneys and brains for determination of virus titers by EID₅₀. The remaining 3 mice were euthanized for lung viral loads by 5 dpi. a Lung viral loads of mice inoculated with H7N9 virus at 3 and 5 dpi. b Viral distribution of H7N9 virus in different tissues. The limit of virus detection is indicated by a dotted line.

Fig. 3.

Histopathology and immunohistochemistry examination of lungs of the parental and mouse-adapted H7N9 viruses. 3 days after the inoculation, the lungs (n = 3) were examined by H&E staining for pathological changes and by anti-influenza nucleoprotein (NP) antibody for detection of antigens (×400). a Histopathology examination. Yellow, blue and red arrows indicated congestion, inflammatory cells infiltration and deciduous cells in the bronchial lumen, respectively. b Immunohistochemistry examination.

Fig. 4.

Growth kinetics of the parental and mouse-adapted H7N9 viruses. MDCK cells were inoculated with H7N9-53, H7N9-53 MA, H7N9-MCX, H7N9-MCX MA, H7N9-ZSM, H7N9-ZSM MA (MOI=0.01), respectively. Supernatants of the cultured cells were collected at a 12-hour interval till 72 hpi. Subsequently, the virus titers were measured by plaque assay and represented by means (± standard deviation).