Comparison of nucleotide sequences of the PB2 gene near positions encoding for amino acid 627 between duck, quail- and chicken-adapted viruses before and after a single round of infection in mice.

<p>Alignment of electropherograms from PB2 sequences obtained from the WT702, QA23, and QA23CkA10 viruses before and after viruses were isolated from the lungs of mice. The arrowhead indicates the position of nucleotide change from G to A that generates the E627K mutation in PB2. E, K, or K/E, on the right of each electropherogram correspond to the predicted encoded amino acid for position 627 in the PB2 open reading frame. PP, sequence derived from plaque purified virus; ML, sequence derived from virus in mouse lung; ML-Egg, sequence derived from virus isolated from mouse lungs and amplified once in embryonated eggs.</p

Comparison of nucleotide changes among the genomes of WT702, QA23 and QA23CkA10 viruses.

*<p>, Indicates synonymous mutations</p>†<p>, indicates nucleotide changes in non-coding region</p

Replication of H9N2 viruses mouse lung.

a<p>Data are the average virus titer from the 4 mice lungs.</p>b<p>, Indicates virus not detected in the 10-fold diluted samples inoculated into eggs.</p><p>NT, Not tested.</p

Quail and Chicken infectious doses of the adapted viruses.

*<p>, Infectious doses were determined from the tracheal swab taken at 3 dpi and expressed in EID50.</p

Replication and transmission of H9N2 viruses in white leghorn chickens.

*<p>, Data from two independent studies.</p>†<p>, Data from four independent studies; NT, Not tested; NA, Not applicable; T and C indicates tracheal and cloacal swabs, respectively.</p

Replication of H9N2 duck, quail- or chicken-adapted viruses.

<p>A) Quail and B) chickens were infected with the indicated doses (in EID₅₀) of WT702, QA23, or QA23CkA10 viruses (set of birds infected with given virus are marked as +++). Each bar corresponds to virus titers in lung homogenates of individual birds collected at 3 dpi. C) Chickens infected with the indicated dose of WT702, QA23, or QA23CkA10 viruses (set of birds infected with given virus are marked as +++). At 3 dpi, chickens were sacrificed and intestine collected to determine virus titers. * indicates no virus detected in the undiluted samples. Virus titers presented as log₁₀EID₅₀/mL.</p

Replication and transmission of H9N2 viruses in Japanese quail.

*<p>, Data from tracheal swabs.</p><p>NA, Not applicable.</p

Comparison of amino acid changes in the internal proteins of WT702, QA23, QA23CkA10 viruses with other influenza viruses from other animal species.

*<p>, Data are from the alignment of 500 sequences available in the influenza sequence database.</p>†<p>, Data are from the alignment of all the sequences available in the influenza sequence database.</p

Body weight changes and lung virus titers in mice infected with H9N2 viruses.

<p>A) Groups of 4 mice were inoculated intranasally with 10⁷ EID₅₀ of QA23ML-3 Egg (black squares) or QA23 PP (open squares) viruses. Body weight and clinical signs of disease were monitored for 14 dpi. Data presented correspond to average body weight changes in each group with corresponding standard deviations. †, one mouse infected with the QA23ML-3 Egg was humanely sacrificed due to severe disease signs. B) Virus titers in lung homogenates of mice (4/group) infected with 10⁷ EID₅₀ of the WT702, QA23CkA10 PP, QA23 PP and QA23ML-3 Egg viruses as indicated. E627 and K627 correspond to the encoded amino acid position 627 in PB2.</p

Plaque morphology and growth kinetics of H9N2 viruses in MDCK and CEK cells.

<p>A) Confluent monolayers of MDCK or CEK cells were infected with the corresponding viruses and maintained at 37°C in agar-maintenance medium supplemented with TPCK-trypsin. Cells were stained with crystal violet at 4 (MDCK) and 2 (CEK) dpi. B) Growth kinetics in confluent monolayer of MDCK (top panels) or CEK (bottom panels) infected with WT702 (open bar), QA23 (shaded bar) or QA23CkA10 (black bar) viruses at an input m.o.i of 0.001. Infected cells were maintained at 37°C in maintenance medium supplemented with TPCK-trypsin. Culture supernatants were collected at different hpi and virus titers measured by HA assay (left panels) or TCID₅₀ in CEK cells (right panels).</p