Table 1.
Genotype of the viruses.
Figure 1.
Eurasian-origin NA and M gene segments contribute to RD transmission of the pH1N1 virus.
Four ferrets were inoculated IN to test the RD transmission of Rec pH1N1 (A) or the 6∶2 reassortant (B) viruses. Nasal washes were collected on the indicated days. Each bar represents the titer of virus from an individual ferret. Inf stands for infected ferret. The limit of detection is represented as the dashed line and is 100.5 TCID50 per mL. Serum was collected on day 0 and day 14. Anti-influenza antibodies were measured by HAI and neutralization assay (C). The limit of detection is 1∶10 for HAI and 1∶20 for the neutralization assay. Antibody titers in the day 0 sera were below the limit of detection.
Figure 2.
Pandemic precursor viruses transmit to 50% of exposed ferrets by RD.
Four ferrets were inoculated IN to test the RD transmission of TRS (A) or the Eurasian (B) viruses. Nasal washes were collected on the indicated days. Each bar represents the titer of virus from an individual ferret. Inf stands for infected ferret. The limit of detection is represented as the dashed line and is 100.5 TCID50 per mL. Serum was collected on day 0 and day 14. Anti-influenza antibodies were measured by HAI and neutralization assay (C). The limit of detection is 1∶10 for HAI and 1∶20 for the neutralization assay. Antibody titers in the day 0 sera were below the limit of detection.
Table 2.
Infectivity of pH1N1 influenza and precursor viruses for ferrets.
Figure 3.
The Eurasian-origin NA and M gene segments contribute to abundant release of large (>4 µm) particles containing influenza virus.
Quantitative (Q)-PCR for influenza A M gene in RNA extracted from the 15 mL collection tube of the cyclone-based air samplers. Air was collected for one hour on the outside of the infected ferret cage. Each bar represents the amount of genome copies of influenza in particles released by a single ferret infected with Rec pH1N1 (A), Eurasian (B), TRS (C), or 6∶2 reassortant (D). Absolute amount of RNA was quantitated using a standard curve of in vitro transcribed influenza M gene RNA. Inf stands for infected ferret.
Figure 4.
The Eurasian-origin NA and M gene segments contribute to the abundant release of 1 to 4 µm particles containing influenza virus.
Q-PCR for influenza A M gene on RNA extracted from the 1.5 mL collection tube of the cyclone-based air samplers. Air was collected for one hour on the outside of the infected ferret cage, each bar represents the amount of particles released by a single ferret infected with Rec pH1N1 (A), Eurasian (B), TRS (C), or 6∶2 reassortant (D). Absolute RNA was quantitated using a standard curve of in vitro transcribed influenza M gene RNA. Inf stands for infected ferret.
Figure 5.
Ferrets infected with the recombinant pH1N1 virus release submicron particles containing influenza virus.
Q-PCR for influenza A M gene on RNA extracted from the filter of the cyclone-based air samplers. Air was collected for one hour on the outside of the infected ferret cage, each bar represents the amount of particles released by a single ferret infected with Rec pH1N1 (A), Eurasian (B), TRS (C), or 6∶2 reassortant (D). Absolute RNA was quantitated using a standard curve of in vitro transcribed influenza M gene RNA. Inf stands for infected ferret.
Figure 6.
Viruses with Eurasian-origin NA have greater neuraminidase activity than viruses with a classical swine NA.
An ELLA assay using fetuin as a substrate was used to determine the NA activity for the biological pH1N1 (•), rec pH1N1 (<$>\vskip -1\scale 70%\raster="rg1"<$>), 6∶2 reassortant (▪), TRS (★), and Eurasian (*) viruses (A). Neuraminidase activity of these viruses was also measured using MUNANA as a substrate (B). Viruses were normalized for equal infectivity in all assays. The data are displayed as an average of 2 independent assays performed in duplicate. Error bars represent the standard error.
Figure 7.
Eurasian-origin gene segments confer filamentous morphology of pH1N1 virus.
Electron micrographs of negatively stained virus preparations are shown for Rec pH1N1 (A), 6∶2 reassortant (B), TRS (C), and Eurasian (D) viruses. Representative images are shown for each virus. Bar; 100 nm.