Abstract
For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3′-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.
Highlights
H9N2 avian influenza A viruses (AIVs) have been reported in human cases, and they act as internal gene donors for the generation of new highly pathogenic, reassortant H5N1 AIVs and low pathogenic H7N9 AIVs that cause poultry as well as human infections [1–6]
Six internal genes (PB1, PB2, PA, NP, M, and NS) of A/Puerto Rico/8/1934 (H1N1) (PR8) and A/chicken/Korea/01310/2001 (H9N2) (E20, 01310) and the 3 end promoter-mutated PB2 and PB1 genes of 01310 (01310-U4) were used in this study [38,39]. 293T, Madin–Darby canine kidney (MDCK) (Madin-Darby Canine Kidney), and A549 cells were purchased from Korean Collection for Type Cultures (KCTC) and maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies Co., Carlsbad, CA, USA) for transfection and the growth kinetics of recombinant viruses
E. coli Toxic open reading frames (ORFs) Is Present in the In-Frame of the HA2 Coding Region of an Egyptian H9N2 Strain
Summary
H9N2 avian influenza A viruses (AIVs) have been reported in human cases, and they act as internal gene donors for the generation of new highly pathogenic, reassortant H5N1 AIVs and low pathogenic H7N9 AIVs that cause poultry as well as human infections [1–6]. Since their first identification in chicken farms in South China in 1994, H9N2 viruses have spread to many countries in Asia and Africa and have differentiated into several (G1-like, Y280/G9-like, and Y439-like) lineages. The evolutionary steps and status of Egyptian H9N2 AIVs with cocirculation of H5N1 AIVs need to be investigated to further understand the potential risk of them becoming pandemic viruses
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