Cross-species spill-over potential of the H9N2 bat influenza A virus

Abstract

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Fig. 1. Phenotypic properties of A/bat/Egypt/381-OP/2017 (H9N2) influenza virus.

A Syncytium formation assay. Monolayers of Vero cells were infected with viruses at an MOI of 3 PFU/cell. At 16 h post infection, cells were treated with TPCK-trypsin, washed, and then treated with pH-adjusted buffers. Cells were then left to recover for 3 h, washed, fixed, and stained for microscopy. Representative images from three independent experiments are shown. B Acid inactivation assay. Viruses were exposed to pH-adjusted buffers at 37 °C for 1 h then neutralized, after which the infectious virus titer was measured by TCID₅₀ assay in MDCK cells. Data from three independent experiments were analyzed with a non-linear regression model by GraphPad Prism, and the calculated virus inactivation pH₅₀ values are shown. C NA activity of human influenza A and B viruses and bat influenza virus as measured by a modified fluorescence-based assay. D Receptor specificity of A/bat/Egypt/381-OP/2017 (H9N2) by glycan microarray. Binding results are presented as bar graphs with bars representing the averaged mean signal derived from six individual replicates of each glycan, with highest and lowest signals removed to give a final average of four median replicates. Error bars represent standard error of the averaged signal. Source data are provided as a Source data file.

Fig. 2. Replication of avian, bat, and human-origin influenza viruses in ex vivo cultures of human bronchus and lung cells and in human airway organoids.

A, C Human bronchial (n = 3 individual donors) and lung tissues (n = 4 individual donors) were infected with A/Hong Kong/415742/2009 (415742pdm), A/Hong Kong/483/1997 (483/H5N1), A/Duck/Hong Kong/Y280/97 (Y280), or A/bat/Egypt/381-OP/2017 (381) at 1 × 10⁶ pfu/mL at 37 °C. Viral titers in culture supernatants collected at 1, 24, and 48 h after infection were determined by TCID₅₀ assays in MDCK cells. B, D The viral loads from (A, C) are depicted as areas-under-the-curve (AUCs). E Human airway organoids (n = 6 individual donors) were infected with the above viruses at 1 × 10⁶ pfu/mL at 37 °C. Viral titers in culture supernatants collected at 1, 24, and 48 h after infection were determined by TCID₅₀ assays in MDCK cells. F Viral titers from (E) are depicted as AUCs. Bar charts show the data as the mean + SD of the results for at least three individual donors. The horizontal line denotes the limit of detection in the TCID₅₀ assay. Statistical analysis was performed using two-way ANOVA with Tukey’s post-test (A, C, E) or one-way ANOVA followed by Tukey’s post-test (B, D, F). P < 0.05 was considered to indicate statistical significance, and exact P values are presented. Source data are provided as a Source data file.

Fig. 3. Virus titers and cytokine and chemokine gene expression detected in the supernatant of infected alveolar epithelial cells.

A Alveolar epithelial cells (AECs) were infected with the A/Hong Kong/415742/2009 (415742pdm), A/Hong Kong/483/1997 (483/H5N1), A/Duck/Hong Kong/Y280/97 H9N2 (Y280), or A/bat/Egypt/381-OP/2017 H9N2 (381) viruses at an MOI of 0.01 and maintained in culture at 37 °C. Viral titers in culture supernatants collected at 1, 24, and 48 h post inoculation (hpi) were determined by TCID₅₀ assays in MDCK cells. Bar charts show the data as the mean +/− SD (n = 6 individual donors). B Viral titers from panel A are depicted as AUCs. Bar charts show the data as the mean + SD (n = 6 individual donors). The horizontal line denotes the limit of detection of the TCID₅₀ assay. C AECs were infected with the indicated viruses at an MOI of 2 and maintained in culture at 37 °C. Expression of the mRNA of viral M genes and of the mRNAs encoding cytokines (IFN-β and IFN-λ1) and chemokines (IP-10; regulated on activation, normal T cell–expressed and secreted [RANTES]; and MCP-1) in AECs at 24 hpi is shown. Bar charts show the data as the mean + SD of the results for five individual donors. Statistical analysis was performed using two-way ANOVA with Tukey’s post-test (A) or one-way ANOVA followed by Tukey’s post-test (B, C); P < 0.05 was considered to indicate statistical significance, and exact P values are presented. Source data are provided as a Source data file.

Fig. 4. Pathogenicity of A/bat/Egypt/381-OP/2017 (H9N2) and A/mallard/Alberta/17/1991 (H9N2) viruses in mice.

Groups of 5- to 6-week-old DBA/2J mice (n = 5) were inoculated i.n. with the indicated doses (10¹, 10², 10³, 10⁴, 10⁵, or 10⁶ EID₅₀) of A/bat/Egypt/381-OP/2017 (H9N2) and A/mallard/Alberta/17/1991 (H9N2) viruses. The mean values +/− SD of body weight loss (A, B) and survival (C, D) were evaluated daily for 14 days. Groups of mice (n = 3) that were infected with 10⁶ EID₅₀ were euthanized at 3 or 5 dpi (E, F), and their lungs and nasal turbinates were harvested, homogenized, and used to quantify the viral titers by EID₅₀ assays. Viral titers expressed as the log₁₀ EID₅₀/ml were plotted as the mean. Statistical analysis was performed using two-way ANOVA (***P < 0.001, ****P < 0.0001). Source data are provided as a Source data file.

Fig. 5. Pathogenicity and replication of A/bat/Egypt/381-OP/2017 and A/mallard/Alberta/17/1991 viruses in ferrets.

A, B Ferrets (n = 3/group) were inoculated i.n. with 10⁶ EID₅₀ of each virus studied. Each ferret was paired with an individual naïve ferret at 24 h post inoculation. Viral titers in nasal washes from individual inoculated ferrets (D; red), direct-contact ferrets (DC; blue), and airborne-contact ferrets (AC; green) on the days post inoculation or post contact were determined and presented as the log₁₀EID₅₀/mL. The mean values +/− SD of body weight loss (C, D) and changes in body temperature (E, F) of individually inoculated (D), direct-contact (DC), and airborne-contact (AC) ferrets up to 12 days post inoculation or post contact and the data are presented as mean values +/− SD using GraphPad Prism. Source data are provided as a Source data file.

Fig. 6. Replication of A/bat/Egypt/381-OP/2017 in ferret tissues.

Ferrets (n = 2) were inoculated with 10⁶ EID₅₀ of virus and then humanely euthanized on days 3 and 5 post inoculation. Tissues were collected, and infectious viral titers were determined by EID₅₀ assays. Each bar represents an individual ferret, and the dotted line indicates the lower limit of virus detection by the assay (1.0 log₁₀EID₅₀/mL). Source data are provided as a Source data file.