Viral segment mimicry for influenza a resistance
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[EMBARGOED UNTIL 12/01/2025] The genomic plasticity of influenza viruses, their sporadic migration, and their rapid mutation rate make this family of viruses a threat to both animal agriculture and public health, as a zoonotic agent. Swine are proposed as a "mixing vessel" as they can have co- infections of swine, avian, and human influenzas, rapidly generating novel zoonotic viruses. Influenza types A, B, C, and D viruses are members of the Orthomyxoviridae family with type A influenzas accounting for significant impacts on humans and many production agriculture species (1-3). Influenza viruses possess a negative-sense, single- stranded RNA genome consisting of eight (influenza A and B) or seven (influenza C and D) distinct segments (3, 4). Viral segment transcription and replication occur in the host nucleus and are dependent on viral RNA-dependent RNA-polymerases (RdRp) which transcribe positive-sense RNA from the negative-sense viral genome, a molecular action that is not possible with host proteins alone (5, 6). Previous groups generated transgenic reporters and reporter cell lines to exploit this unique and critical viral action (7-13). The reporter cell line approaches rely on negative-sense RNAs transcribed by RNA Polymerase I (Pol I), flanked by viral regions critical for transcription/replication, effectively mimicking a nascent, naked viral genomic RNA. These "mimic RNAs" remain unexpressed until exposed to the viral transcriptional proteins at which point, viral transcription leads to translation of the reporter protein(s). These reporter lines have allowed for studies on viral replication, evaluation of neutralizing antibodies and antiviral drugs, and viral titration (11-15). The low background expression of these reporters is due to the absence of functional RNA-dependent RNA polymerases in vertebrates (16). These reporter cell lines represent an autonomous, conditionally-expressed reporter strategy with translation only occurring in influenza-infected cells. This strategy may be leveraged to develop cell lines with enhanced antiviral action by replacing reporter genes with genes that enhance antiviral action following viral infection. Enhanced apoptosis is suggested as a host defensive strategy to limit the impact of an invading pathogen (17-19). A biological example has been reported in porcine cells infected with SARS-CoV-2 (19). These infected cells enhance apoptosis, limiting viral production and protecting the individual from systemic disease. RNA Pol I transcribed viral reporters are functional in cell culture, however, no functional Pol I-driven transgenic animals have been described. RNA polymerase II promoters (Pol II) are suitable for generating functional transgenic animals (20-22). However, Pol II transcripts have different termini than viral genomes (9, 23). We predicted that a Pol II promoter could be modified to transcribe a suitable mimic reporter. We also predicted that if the reporter gene were exchanged for a lethal gene, the viral-mediated expression would lead to reduced viability of the infected cells, disrupting infection progression, reducing viral production, and resulting in individuals that are resistant to wide-scale infection.
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Ph. D.