Table 1.
Design strategies for IAV genomes that propagate 9 genomic segments.
A description of the manipulated packaging signals, encoded proteins, and success of rescuing each 9 segmented IAV strategy.
Fig 1.
Viruses that require 9 genomic segments for infectivity generate more defective particles than the parental strain.
(A and B) Potential genomic configurations of the 9S PB1 mCherry virus (A) and the 9S PB2 sfGFP virus (B) assuming packaging of at least 8 segments. (C) Fluorescent microscopy images of 9S PB1 mCherry, 9S PB2 sfGFP, or WT PR8 virus-infected MDCK cells at 6, 12, and 24 hours post-infection at an MOI of 0.1; nuclei were stained blue using DAPI staining and the scale bar represents 100 micrometers. (D and E) qRT-PCR of specific vRNA segments of the 9S PB1 mCherry virus (D) and the 9S sfGFP virus (E) after growth in eggs normalized to wild-type PR8 segment levels. Artificial segments were normalized based on the packaging signals of that segment (i.e. the PB1 mCherry segment was normalized to wild-type PB1). (F) Multicycle-growth curve of 9S PB1 mCherry, 9S PB2 sfGFP, and WT PR8 viruses in MDCK cells (MOI = 0.05). (G) Endpoint titer 72 hours post-infection in 10-day old embryonated chicken eggs, Madin-Darby canine kidney cells and A549 human lung-epithelial cells of the indicated viruses (MOI = 0.05). (H) HA assays 72 hours post-infection in 10-day old embryonated chicken eggs, Madin-Darby canine kidney cells and A549 human lung-epithelial cells of the indicated. (I) The “DI Units” of the 9-segmented fluorescent viruses as compared to that of WT PR8 virus, calculated by dividing respective normalized HA units by normalized endpoint titer. For all graphs, * represents a p-value of ≤ .05 and ** represents a p-value of ≤ .001.
Fig 2.
Viruses that require 9 genomic segments are highly attenuated and can interfere with lethal viral challenge.
(A–C) Weight loss curves from infections with the indicated doses of WT PR8 virus (A), the 9S PB1 mCherry virus (B), or the 9S PB2 sfGFP virus (C). (D-F) Survival curves from infections with the indicated doses of WT PR8 virus (D), the 9S PB1 mCherry virus (E), or the 9S PB2 sfGFP virus (F). (G) Experimental design of the coinfection challenges in C57BL/6J mice. (H) Weight-loss curves from infecting mice with a lethal dose of WT PR8 (20 PFU), a sublethal dose of the 9S PB1 mCherry virus (500 PFU), a sublethal dose of the 9S PB2 sfGFP virus (500 PFU) a combination of WT PR8 and the 9S PB1 mCherry virus, or a combination of WT PR8 and the 9S PB2 sfGFP virus. (I) Survival curves from challenges described in H.
Fig 3.
Influenza viruses can harbor and propagate an artificial, defective interfering-like genomic segment.
(A) A schematic comparing the 9S PB1-mCherry-PB1 segment and the INS002 PB1 DI segment, which acted as a basis for the design of the 9S PB1 DI segment. (B) Potential genomic configurations of the 9S PB1 DI virus assuming packaging of at least 8 genomic segments. (C) qRT-PCR of specific vRNA segments of the 9S PB1 DI virus after growth in eggs normalized to wild-type PR8 segment levels. Segments were normalized based on the intact packaging signals of that segment (i.e. the PB1 DI segment was normalized to wild-type PB1) (D) Multicycle growth curve of the 9S PB1 DI virus on MDCK-cells infected at an MOI of 0.05. (E) Endpoint titer 72 hours post-infection in embryonated chicken eggs, Madin-Darby canine kidney cells (MOI = 0.05), and A549 human lung-epithelial cells (MOI = 0.05) of the 9S PB1 DI and WT PR8 virus. (F) HA assay 72 hours post-infection in 10-day old embryonated chicken eggs, Madin-Darby canine kidney cells and A549 human lung-epithelial cells of the 9S PB1 DI virus and WT PR8 virus. (G) “DI Units” of the viruses were calculated by dividing HA units by endpoint titer. (H) Mouse bodyweight curves after infection with the indicated doses of the 9S PB1 DI virus. (I) Survival curves from H. (J) Mouse bodyweight curves after infecting mice with a sublethal dose of the 9S PB1 DI virus (500 PFU), a lethal dose of WT PR8 (20 PFU), or combination of WT PR8 and the 9S PB1 DI virus. (K) Survival curves from the infections in J. For all graphs, * represents a p-value of ≤ .05 and ** represents a p-value of ≤ .001.
Fig 4.
Viruses that require 10-segments for infectivity are viable and generate large amounts of DI particles.
(A) Potential genomic configurations of the 10S virus assuming packaging of at least 8 genomic segments. (B) Fluorescent microscopy images of 10S or WT PR8 virus-infected MDCK cells at 6, 12, and 24 hours post-infection at an MOI of 0.1; nuclei were stained blue using DAPI staining, and the scale bar represents 100 micrometers. (C) Growth curve of the 10S and parental PR8 virus in MDCK cells infected at an MOI of 0.01. (D) qRT-PCR for the indicated genomic segments from purified 10S virions. (E) Endpoint titer 72 hours post-infection in 10-day old embryonated chicken eggs, Madin-Darby canine kidney cells (MOI = 0.01) and A549 human lung-epithelial cells (MOI = 0.01) of the 10S and WT PR8 virus. (E) HA assay of the 10S and WT PR8 virus 72 hours post-infection in 10-day old embryonated chicken eggs, Madin-Darby canine kidney cells and A549 human lung-epithelial cells. (F) The “DI Units” of the viruses were calculated by dividing HA units by endpoint titer. For all graphs, * represents a p-value of ≤ .05 and ** represents a p-value of ≤ .001.
Fig 5.
Influenza viruses that require 10 genomic segments for infectivity are highly attenuated and their administration protects mice from wild-type viral challenge.
(A) Weight loss curves from infections with the indicated doses of 10S virus. (B) Survival curves from infections described in A. (C) Diagram detailing the coinfection challenge experimental design in C57BL/6J mice. (D) Bodyweight curves after infecting mice with a sublethal dose of the 10S virus (5000 PFU), a lethal dose of WT PR8 (20 PFU), or a combination of the WT PR8 and 10S viruses. (E) Survival curves from the infection groups described in D.
Fig 6.
Viruses that require 10 genomic segments reassort with, and restrict the growth of, wild-type virus.
(A-B) Weightloss (A) and survival (B) curves of mice treated with 5x103 PFU of the 10S therapeutic virus and infected with either 20, 50, or 500 PFU of WT PR8. For panel B, the significance of the 20 and 50 PFU challenge dose was the same. (C) Viral titering of IAV from lungs of mock infected mice, mice 4 days post-infection with 5x103 PFU of 10S virus alone, 20 PFU of WT PR8 alone, or coinfected with WT PR8 and the 10S virus. (D) Number of plaques assayed for fluorescence from each of the treatment group lung homogenates from C. ND-Not detected, NA-Not applicable. (E) RT-PCR and sequencing analysis of a plaque-purified virus that displayed only red fluorescence isolated from the WT PR8/10S-treated group from C. Multiple independently isolated viruses displayed the same genetic makeup. (F) Schematic of the inferred sources of the genomic segments detected in the analysis described in E. For all graphs, * represents a p-value of ≤ .05.
Fig 7.
Influenza viruses that require 10 genomic segments can interfere with a lethal viral challenge when administered therapeutically.
(A) Diagram of the 10S therapeutic administration experiment in C57BL/6 mice, with treatment at 24 hours post-infection with a lethal dose of WT PR8. (B) Bodyweight curves of mice treated with a dose of the 10S virus (5000 PFU), infected with a lethal dose of WT PR8 (20 PFU), or infected with WT PR8 virus followed by 10S virus administration 24 hours later. (C) Survival curves from B.