Diagrams of chimeric constructs and recombinant HEF viruses. (A) Construction of the NA-NP-NA chimera. This construct contains the PR8 NA packaging sequences in the flanking regions and the mutated PR8 NP ORF (NPmut) in the middle. The hatched boxes represent serial silent mutations introduced into the terminal ORF regions. The ATGs in the 3′ NA ORF packaging sequence region were all mutated to TTGs (). (B) Rewired RNA segments used for generating recombinant viruses. All these constructs carry the NA or HA packaging sequences in the flanking regions and the mutated ORFs in the center. Only the HEF ORF of the NA-HEF-NA and HA-HEF-HA constructs does not carry serial silent mutations at the two ends. In these constructs, the 3′-end NA and HA packaging sequences are not translated due to the removal of ATGs. The hatched boxes also represent serial silent mutations introduced into the terminal ORF regions for each construct. (C) GFP constructs carrying segment-specific influenza A virus packaging signals. All of the constructs carry a GFP gene in the middle and influenza PR8 virus vRNA packaging sequences at the two ends. The ATGs in the 3′-end packaging sequences were removed by silent mutations. (D) Diagrams of a 7-segmented HEF virus (ΔPB2-PS) and an 8-segmented HEF GFP virus (ΔPB2-PS+PB2-GFP-PB2). ΔPB2-PS includes two chimeric segments (NA-PB2-NA and HA-HEF-HA), shown in panel B, and five wild-type PR8 virus segments (PB1, PA, NP, M, and NS). The ΔPB2-PS+PB2-GFP-PB2 virus contains an eighth GFP segment (PB2-GFP-PB2), shown in panel C. The remaining 7-segmented and 8-segmented viruses listed in were generated in a similar fashion.

Immunofluorescence of 293T cells infected with 7-segmented and 8-segmented HEF viruses. The rescued HEF viruses were passaged in embryonated eggs two or three times to reach peak titers. 293T cells in 24-well plates treated with poly-d-lysine were then infected with undiluted 7- or 8-segmented HEF viruses—ΔPB2-PS and ΔPB2-PS+PB2-GFP-PB2 (A), ΔPA-PS and ΔPA-PS+PA-GFP-PA (B), ΔNP-PS and ΔNP-PS+NP-GFP-NP (C), ΔM-PS and ΔM-PS+M-GFP-M (D), ΔPB1-PS and ΔPB1-PS+PB1-GFP-PB1 (E), ΔHA-PS and ΔHA-PS+HA-GFP-HA (F), and ΔNS-PS and ΔNS-PS+NS-GFP-NS (G)—and incubated in a 37°C incubator overnight. The cells were then visualized for NP (red) and GFP expression by using an immunofluorescence assay (see Materials and Methods). All the GFP viruses (A to G) and the three 7-segmented viruses ΔPB1-PS (E), ΔHA-PS (F), and ΔNS-PS (G) were normalized based on the hemagglutination assay titers measured by using chicken red blood cells, and the multiplicity of infection for these viruses was around 5 to 10; for the remaining 7-segmented viruses, those with the highest TCID₅₀/ml after passages in eggs were used to infect 293T cells.

Growth of HEF viruses in embryonated chicken eggs. Eight-day-old chicken eggs were inoculated with 100 TCID₅₀ of ΔPB2-PS and ΔPB2-PS+PB2-GFP-PB2 (A), ΔPA-PS and ΔPA-PS+PA-GFP-PA (B), ΔNP-PS and ΔNP-PS+NP-GFP-NP (C), ΔM-PS and ΔM-PS+M-GFP-M (D), ΔPB1-PS and ΔPB1-PS+PB1-GFP-PB1 (E), ΔHA-PS and ΔHA-PS+HA-GFP-HA (F), and ΔNS-PS and ΔNS-PS+NS-GFP-NS (G) viruses; incubated at 33°C for 3 days; and transferred to 4°C overnight. The allantoic fluids were then harvested, and the virus titers (TCID₅₀/ml) were determined in 96-well plates using an immunofluorescence method with anti-PR8 NP mouse monoclonal antibody HT103 () and Alexa Fluor 594-labeled donkey anti-mouse IgG (Invitrogen) (). Three viruses, ΔPB2-PS (A), ΔNP-PS (C), and ΔM-PS (D), grew very poorly and were easily lost during the passages. Therefore, we could not determine accurate titers for them. We estimated their titers at less than 10³, 10², and 10² TCID₅₀/ml, respectively. The mean titer ± standard deviation calculated from three eggs is shown for each virus.

Genome compositions of four HEF GFP viruses. (A) The PR8 virus was grown in 10-day-old chicken eggs at 37°C for 2 days. (B to E) The HEF viruses ΔPB2-PS+PB2-GFP-PB2 (B), ΔPA-PS+PA-GFP-PA (C), ΔNP-PS+NP-GFP-NP (D), and ΔM-PS+M-GFP-M (E) were grown in 8-day-old chicken eggs at 33°C for 3 days. The viruses were then purified by passing them through a sucrose cushion, and RNA was isolated using TRIzol (Invitrogen). The purified RNA was separated on a 2.8% denaturing polyacrylamide gel containing 7 M urea. The gel was stained with a SilverXpress silver-staining kit (Invitrogen) to visualize the RNA bands. The bands representing the genomic segments of the virus were labeled for each gel. The two bands corresponding to the M (or rewired M [E]) segment and the NS segment (labeled “a” and “b,” respectively) were quantified for intensity using ImageQuant software. The graphs used to quantify the two peaks (a and b) are shown at the lower right of each gel. The numbers represent the relative peak volumes (with peak b set to 1). Note that the gels shown were run at different times.

Genome compositions of recombinant 7- and 8-segmented HEF viruses carrying a rewired PB1, HA, or NS segment. Using the same method as in , six HEF viruses, ΔPB1-PS (A), ΔPB1-PS+PB1-GFP-PB1 (B), ΔHA-PS (C), ΔHA-PS+HA-GFP-HA (D), ΔNS-PS (E), and ΔNS-PS+NS-GFP-NS (F), were analyzed for genome composition using an RNA gel and silver-staining method. The quantifications of the bands at the bottom of each gel are also shown. ?, unknown band.

qRT-PCR analysis of recombinant HEF GFP viruses. (A) Procedure for the experiment. (B) GFP/NP vRNA ratios for each virus. In total, seven viruses were analyzed: ΔPB2-PS+PB2-GFP-PB2, ΔPA-PS+PA-GFP-PA, ΔNP-PS+NP-GFP-NP, ΔM-PS+M-GFP-M, ΔPB1-PS+PB1-GFP-PB1, ΔHA-PS+HA-GFP-HA, and ΔNS-PS+NS-GFP-NS. The GFP/NP vRNA ratio for the ΔNP-PS+NP-GFP-NP virus was arbitrarily set at 1. The experiment was performed in triplicate, and the mean value ± standard deviation is shown for each virus.

smFISH analysis of single virus particles. The smFISH experiment (see Materials and Methods) was performed to measure the percentages of GFP and NP vRNA colocalized particles. (A) Experimental procedure. (B) Percentages of GFP and NP colocalized particles. Two viruses, ΔPA-PS+PA-GFP-PA and ΔPB1-PS+PB1-GFP-PB1, were analyzed. For each virus, at least 10 images were analyzed, and the mean values ± standard deviations are shown. On average, 500 to 700 particles were counted for each image.