Table 1.
Isolates used in the study.
Table 2.
Primers used for PCR.
Fig 1.
CPE development in CHSE-214 cells after infection with different aquabirnavirus isolates.
Phase-contrast microscopical images at different times post infection.
Fig 2.
Replication curves for different aquabirnaviruses in CHSE-214 cells.
Cell monolayers were infected with different IPNV isolates at 20pfu/cell. Amounts of virus in A) intracellular or B) supernatant at different time points post infections were determined by titration in CHSE-214 cells. The data are presented as mean ±S.E.M. log10 TCID50/ml of minimum of 5 replicated taken from at least two independent experiments.
Fig 3.
Impact of different aquabirnavirus isolates on global protein synthesis in CHSE-214 cells.
Cells infected with either E1S, VR-299, TA or PT isolates collected at different times post infection were incubated with S35 Methionine for 30 min before lysis. Control cells were used in addition. Lysates (7μg total proteins) were subjected to SDS-PAGE, autoradiographed by incubation in storage phosphor cassettes before analysis by Typhoon. The ImageQuant software (GE Healthcare) was used to estimate the protein quantity by measuring the density of one band at different time points for each isolate. A) Autoradiograph image showing virus (VP1-4) and host protein (arrow) bands as an example. C = control sample at 48hrs post infection; Percentages = relative amounts of S35 Methionine-labeled host cell proteins calculated on the basis of the protein band shown as an arrow. B) Histogram of S35 Methionine-labeled host cell proteins of infected relative to uninfected cells as described above. Each entry represents three independent experiments and error bars are ± S.E.M. Key: VR = VR299.
Fig 4.
Type I IFN response induced by different isolates of aquabirnaviruses.
Normalized gene expression of A) IFNα, B) PKR C) Mx1, and D) TNFα2 at different time points post infection ± S.E.M.. *P<0.05; **P<0.01; ***P<0.001. N = 3.
Fig 5.
Cell death induced by different virus isolates of aquabirnaviruses.
Flow cytometric analysis of FITC-Annexin (a) and PI (b) staining of cells infected with different isolates. The charts show the percentage of FITC-annexin stained cells (apoptosis) and PI stained cells (cells with compromised membranes) after subtracting the percentage of the FITC-annexin and PI positive percentages obtained from uninfected cells, respectively. The data represent the means of three independent experiments ±SD.