Protease activation mutants elicit protective immunity against highly pathogenic avian influenza viruses of subtype H7 in chickens and mice

Abstract

Protease activation mutants of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) have been generated that are fully dependent on the presence of trypsin for growth in cell culture. Unlike wild-type virus, the mutants do not induce systemic infection in chicken embryos and show low pathogenicity in both chicken embryos and adult chickens. Inactivated vaccines prepared from the mutants protected chickens and mice very efficiently against infection with highly pathogenic wild-type virus in a cross-reactive manner. The potential of these mutants to be used as veterinary and prepandemic vaccines will be discussed.

Cholesterol binding to the transmembrane region of a group 2 HA of Influenza virus is essential for virus replication affecting both virus assembly and HA’s fusion activity

“…To investigate whether cholesterol binding to HA affects virus replication, we created the described mutations in the CCM of HA in the context of the viral genome. We used a variant of fowl plague virus (A/FPV/Rostock/34, H7N1), termed FPV* which contains a monobasic cleavage site in HA and thus requires trypsin for growth in cell culture (38). The amino acid exchanges were generated by at least three nucleotide substitutions to exclude that mutant viruses revert back to wild type.…”

“…Mutant 1 of the highly pathogenic strain A/FPV/Rostock/1934 (H7N1), termed FPV*, that contains the sequence PSKGR instead of PSKKRKKR at the C-terminus of HA1 (38) was used to create recombinant virus. FPV* shows low pathogenicity in chicken and requires trypsin for growth in cell culture and is thus suitable for working in a BSL2 lab.…”

“…The full-length sequence (excluding the fluorophore) of HA mutants LA, YK2A, LW2A and YKLW4A were cloned from plasmid pECerulean (37) to pHH21 with In-Fusion® HD Cloning Kit (Takara Bio, Japan). Recombinant Influenza viruses were produced with the twelve plasmids system (38) by transfection (0.5 µg of each plasmid) of 293T cells in 35 mm dishes with TurboFect reagent. 4-6 h later, the medium was changed to infection medium (DMEM, 0.1% FCS, 0.2% BSA, 1 µg/ml TPCK-Trypsin).…”

Mimicking the passage of avian influenza viruses through the gastrointestinal tract of chickens

“…8E11 and Madin Darby canine kidney (MDCK II) cells were grown in DMEM (Dulbecco's modification of Eagle's medium, PAN, Aidenbach, Germany) supplemented with 10% FCS (fetal calf serum, Perbio, Bonn, Germany) and penicillin [100 U/ml]/ streptomycin [100 μg/ml] at 37°C and 5% CO 2 . Mutant 1 (M1) of the highly pathogenic strain A/FPV/Rostock/1934 (H7N1) was used in the experiments, that contains the sequence PSKGR instead of PSKKRKKR at the C-terminus of HA 1 (Wagner et al, 2013). This mutation creates a low pathogenic strain suitable for working in a BSL2 laboratory.…”

“…PSKKRKKR is mutated to PSKGR. Unlike FPV with a polybasic cleavage site, FPV M1 shows low pathogenicity in chicken and requires trypsin for growth in cell culture (Wagner et al, 2013). For the following experiments, we concentrated FPV M1 by ultracentrifugation from the supernatant of cells, which were infected at a high MOI and incubated for ∼12 h in the absence of trypsin.…”