2.2. Reverse Genetics (Rg) Systems for H5N1EGY and H9N2EGY and Generation of Reassortant, Mutant and Wild-Type Strains

To generate wild-type strains and H5N1-subtype reassortants with specific genetic constellations (Figure 1), 8 μg of plasmid DNA (1 μg of each plasmid) encoding different combinations of the eight viral segments were transfected into a co-culture of 293T/MDCK-II cells (ratio 3:1) as previously described [14,17,18]. The transfection mixture was harvested 72 h post-transfection (p.t.). For LPAIV H9N2_EGY, 1 µg/mL of L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin was added to the transfection mixture 24 h p.t. and the transfection mixture was then harvested 48 h after addition of TPCK-treated trypsin. An aliquot of 100 µL of each supernatant was used to inoculate SPF embryonated eggs for another 48 h. The harvested viruses of compatible genetic constellations (Figure 1), were titrated using focus forming assay, titred, aliquoted and stored at −80 °C for further use.

Genetic compatibility between Egyptian H5N1- and H9N2-viruses. The six internal-proteins encoding segments of LPAIV H9N2_EGY, were placed individually and in combination, into the genetic background of HPAIV H5N1_EGY. The gene segments from H5N1_EGY and H9N2_EGY are colored in grey and red, respectively. The six internal-proteins encoding segments from H7N9_Anhui (control) are colored in green. All genetic constellations used in this study were transfected to co-culture of 293T/MDCK-II cells and the rescued wild-type, reassortant and mutant viruses were propagated on embryonated SPF eggs. All genetic combinations were compatible showing variable hemagglutination unit (HAU) and focus forming unit (FFU) titers.

Mutations of interest were introduced into the corresponding PA genes by using QuikChange site-directed mutagenesis (Agilent, Santa Clara, CA, USA) and specific mutagenesis primers (Table 1) according to manufacturer instructions. Sanger sequencing was performed to confirm the introduction of these mutants. Variant viruses H5N1_{EGY_R367K} and H5N1_{PA-H9N2Egy_K367R} carrying polymerase acidic (PA)-encoding segments with specific mutations were rescued individually by transfecting a co-culture of 293T/MDCK cells with eight pMPccdB plasmids corresponding to the unchanged seven segments of the virus and the mutated one (7 WT viral segments + 1 mutant PA viral segment), using Trans-IT2020 (Mirus Bio, Madison, WI, USA) as described previously [14,19]. Recombinant viruses were rescued as described above and were subsequently sequenced to confirm the correct variant PB2 and PA segment in the genetic backbone of the other seven segments. Stocks of viruses harvested from infected MDCK-II cells were titrated by focus forming assay and kept at −80 °C until further use.

Site directed mutagenesis primers.