Figure 1.
Generation of viruses relevant to Bat09.
(A) EM picture of Bat09 (left), Bat09:mH1mN1 (middle), and PR8 (right). (B) Viral titers in transfection supernatants of Bat09 and PR8-HA/NA reassortants, and Bat09:mH1mN1. Each bar represents an independent rescue experiment. (C) The mH1 contains PR8-HA coding region and Bat09-HA packaging region (start codon removed). The mN1 was constructed using the same strategy. (See Fig. S2A for details.) (D) Bat09:mH1mN1 and PR8 replication kinetics in MDCK cells. (E) Peak titers of the viruses in embryonated chicken eggs. *, P<0.05, compared to PR8.
Figure 2.
Pathogenicity of Bat09:mH1mN1 and PR8 viruses in mice.
(A) Virus titers of Bat09:mH1mN1 and PR8 in mouse lungs at 3 and 5 dpi. Each mouse was intranasally inoculated with 103 TCID50 of each virus. (B) Mouse weight on each day post inoculation was represented as a percentage of the weight on day 0 (100%). Each mouse was intranasally inoculated with 104 TCID50 of Bat09:mH1mN1 or PR8. (C) Survival rate of the mice inoculated with 104 TCID50 of virus. (D) H&E staining of microscopic lung sections from mice inoculated with 103 TCID50 of virus at 5 dpi. (E) IHC staining of lung sections at 5 dpi. *, P<0.05, Bat09:mH1mN1 compared to PR8.
Figure 3.
Diagram of the Bat09:mH3mN2 construct, and pathogenicity of Bat09:mH3mN2 and TX98 viruses in mice.
(A) Modified HA (mH3) and modified NA (mN2). To construct the mH3, TX98-HA coding region was flanked by the putative packaging regions from Bat09-HA and all potential start codons in the Bat09-HA 5′ packaging region were mutated. To construct the mN1, PR8-NA coding region was flanked by the putative packaging regions from Bat09-NA and all start codons in the Bat09-NA 5′ packaging region were mutated. (B) Bat09:mH3mN2 and TX98 replication kinetics in MDCK cells. MDCK cells were inoculated at a multiplicity of infection (MOI) of 0.01 TCID50/cell with the Bat09:mH3mN2 or TX98 viruses. (C) Weight loss of mice mock-infected or infected with Bat09:mH3mN2 or TX98 viruses. Each mouse was intranasally inoculated with 3×105 TCID50 of each virus. (D) Virus titers of Bat09:mH3mN2 and TX98 viruses in mouse lungs at 3 and 5 dpi. Each mouse was intranasally inoculated with 3×104 TCID50 of each virus. *, P<0.05, Bat09:mH3mN2 compared to TX98.
Figure 4.
In vitro property of Bat-NS1 mutants.
(A) Luciferase reporter mediated assay to quantitate the NS1 protein inhibition effects on interferon-β promoter activation. (B) VSV-luciferase mediated bioassay to quantitate the inhibitory effects on VSV virus infection resulted from the immune response induced by the different NS1 WT or truncated viruses. (C) Virus replication kinetics in human lung epithelial Calu-3 cells. * or #, P<0.05, Bat-NS1-128 compared to PR8-NS1-126 (*) and Bat-NS1-73 compared to PR8-NS1-73 (#) are shown in (B) and (C).
Figure 5.
Pathogenicity of Bat-NS1 mutants in mice.
(A) Virus titers of Bat09:mH1mN1ss and NS1 mutants in mouse lungs at 3 and 5 dpi. Each mouse was intranasally inoculated with 104 TCID50 of each virus. (B) Mouse weight on each day post inoculation was represented as a percentage of the weight on day 0 (100%). Each mouse was intranasally inoculated with 105 TCID50 of the indicated viruses. (C) Survival rate of the mice inoculated with 105 TCID50 of the viruses. Higher virus doses were used in this experiment based on the PR8-NS1-126 control virus, which caused significant weight loss but had low mortality at 105 TCID50 so the attenuation of Bat-NS1 truncated viruses can be appropriately compared to it. *, P<0.05, truncated Bat09:mH1mN1ss-128 and Bat09:mH1mN1ss-73 compared to PR8-NS1-126.
Figure 6.
Pathogenicity of Bat-PB2 mutants in mice.
(A) Virus titers of Bat09:mH1mN1 and PB2 mutants in mouse lungs at 3 and 5 dpi. Each mouse was intranasally inoculated with 103 TCID50 of each virus. (B) Mouse weight on each day post inoculation was represented as a percentage of the weight on day 0 (100%). Each mouse was intranasally inoculated with 104 TCID50 of the indicated viruses. (C) Survival rate of the mice inoculated with 104 TCID50 of the viruses. *, P<0.05, PB2 mutants compared to Bat09:mH1mN1. For mouse body weight, * is only marked on the first day of each group that is significantly different from Bat09:mH1mN1.
Figure 7.
Polymerase activity of Bat09 with wild type and mutant PB2.
A luciferase mediated mini-genome replication assay was performed at 33°C, 37°C, and 39°C by co-transfecting 293T cells with Bat09 PB2 (WT or mutant), PB1, PA, NP, and a vRNA-like luciferase reporter. Relative luciferase activity were determined to represent the viral polymerase activity. *, P<0.05, compared to WT.
Figure 8.
Compatibility of the PB2, PB1, PA and NP originated from Bat09 and other influenza viruses.
A mini-genome replication assay was used to determine the compatibility of the different RdRp components. (A–I) PB2, PB1, PA, and NP from Bat09 and various influenza A viruses as indicated. (J) PB2, PB1, PA and NP from Bat09 and B/Russia/69. (K) PB2, PB1, PA and NP from Bat09 and Bat10. The vRNA reporters used for the compatibility test between Bat09 and IAVs (Fig. 8A–I) were an equal ratio of pPolI-Bat-NS-Luc and pPolI-FluA-NS-Luc. For compatibility test between Bat09 and IBV (Fig. 8J) the vRNA reporters used were pPolI-Bat-NS-Luc and pPolI-FluB-NS-Luc. For compatibility test between Bat09 and Bat10 (Fig. 8K) only the pPolI-Bat-NS-Luc plasmid was used.
Table 1.
Rescue efficiency of PB2, PB1, PA reassortants between Bat09:mH1mN1 and PR8.
Table 2.
Rescue efficiency of internal gene reassortants between Bat09:mH1mN1 and PR8.
Table 3.
Rescue efficiency of reassortants with NP and NS containing modified packaging signals.
Table 4.
Rescue efficiency of reassortants containing HA and NA packaging single from other viruses.
Table 5.
Rescue efficiency of reassortants between Bat09:mH1mN1ss and Bat10:mH1mH1ss.