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The 1918 “Spanish Flu” pandemic was cause by an Influenza A virus that infected 500 million people and nearly 50 million people died. Influenza viruses utilize a RNA-Dependant RNA Polymerase (RdRp) complex, composed of the PA, PB1 and PB2 proteins along with the viral nucleoprotein (NP) to mediate viral transcription and replication. The 1918 PB1 gene has been linked to increased virulence in mice and ferrets. We have investigated the role of PB1 in the transcription and replication of the 1918 virus and its relation to pathogenicity by comparing its RdRp to the RdRp of a low virulence conventional H1N1 human virus isolate, A/Canada/RV733/2007 (RV733). Contrary to previous studies, our dual-luciferase reporter assay revealed the 1918 RdRp had lower transcriptional activity than the RV733 RdRp in vitro. The 1918 NP seems to be the key determinant for the difference in transcriptional activity of the 1918 and RV733 RdRp complexes. The 1918 PB1 in the RV733 RdRp maintained high reporter expression while the RV733 PB1 in the 1918 RdRp abolished reporter expression. 1918/RV733 chimeric PB1 proteins were also generated and evaluated with the reporter assay. Recombinant RV733/1918 viruses were generated by reverse genetics and we determined that PB1 is a key determinant of the high growth phenotype of the 1918 virus, but only a minor contributor to pathogenicity. A novel role for the 1918 NP in the high growth phenotype and pathogenicity of the 1918 virus is also described.

The 1918 “Spanish Flu” pandemic was cause by an Influenza A virus that infected 500 million people and nearly 50 million people died. Influenza viruses utilize a RNA-Dependant RNA Polymerase (RdRp) complex, composed of the PA, PB1 and PB2 proteins along with the viral nucleoprotein (NP) to mediate viral transcription and replication. The 1918 PB1 gene has been linked to increased virulence in mice and ferrets. We have investigated the role of PB1 in the transcription and replication of the 1918 virus and its relation to pathogenicity by comparing its RdRp to the RdRp of a low virulence conventional H1N1 human virus isolate, A/Canada/RV733/2007 (RV733). Contrary to previous studies, our dual-luciferase reporter assay revealed the 1918 RdRp had lower transcriptional activity than the RV733 RdRp in vitro. The 1918 NP seems to be the key determinant for the difference in transcriptional activity of the 1918 and RV733 RdRp complexes. The 1918 PB1 in the RV733 RdRp maintained high reporter expression while the RV733 PB1 in the 1918 RdRp abolished reporter expression. 1918/RV733 chimeric PB1 proteins were also generated and evaluated with the reporter assay. Recombinant RV733/1918 viruses were generated by reverse genetics and we determined that PB1 is a key determinant of the high growth phenotype of the 1918 virus, but only a minor contributor to pathogenicity. A novel role for the 1918 NP in the high growth phenotype and pathogenicity of the 1918 virus is also described.

The Mononegavirales order unifies the non-segmented negative sense viruses (nsNSVs), including Marburgvirus (MARV) of the Filoviridae family and respiratory syncytial virus (RSV) of the Pneumoviridae. The mechanism of action of these viruses and how they infect cells are very similar, especially when focusing on their polymerases, which are distinct from those of eukaryotes and therefore possible targets for antiviral drugs. nsNSVs utilize a RNA-dependent RNA polymerase to either replicate the viral RNA genome or transcribe it into positive sense mRNA. Despite this, these two viruses result in very different, but equally devastating, effects in humans. Whereas MARV virus often results in rare but fatal hemorrhagic fevers, RSV is a common seasonal virus that can result in long term negative effects to respiratory health. These negative effects on public health demand extensive research in these two fields and a need to develop new technology and methods in order to uncover the missing pieces of viral gene expression. Specifically, the development of a MARV minigenome system would allow for increased testing of this virus outside of the confines of the biosafety level 4 (BSL-4) setting. By replacing MARV genes with reporter genes, but retaining the characteristic leader, intergenic, and trailer regions of the genome, tests involving site directed mutagenesis would reveal new insights into the crucial genomic elements needed for successful gene expression. Coupled with the transfection of the minigenome with plasmids coding for the crucial MARV proteins, artificial changes to the genome would lead to the presence of absence of translated bioluminescent reporter proteins. Using these two viruses, this study attempted to find commonalities across families. Specifically, the goals of this research were twofold, to find the optimal ratio of MARV plasmids in the minigenome system to understand the effects of the stem-loop secondary structure of MARV mRNA transcripts as well as determine the tail length of the poly(A) tail of RSV mRNA transcripts using digestion and probing primers. Calculating the RSV poly(A) tail length would allow for further research into determining whether the MARV and RSV polymerase polyadenylates before or after it releases the transcript. Despite multiple failed attempts, transfections using pCAGGS plasmids and the eGFP monocistronic minigenome in a 6-well plate qualitatively demonstrated the need for pCAGGS-L plasmid concentration of 1000 ng/µl. Due to time constraints, the poly(A) tail length of the RSV NS-1 mRNA transcript could not be determined. Overall, this study focused on gaining new insights on the techniques and procedures necessary for conducting virus research in a biosafety level 2 (BSL-2) setting, as well as developing troubleshooting skills in approaching fail experiments.