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Research Project: Viral Ecology of Henipaviruses in Endemic Settings and Intervention Strategies to Prevent their Spread to Domestic Animals

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Title: Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses

Author
item DAVIES, KATHERINE - Oak Ridge Institute For Science And Education (ORISE)
item WELCH, STEPHEN - Centers For Disease Control And Prevention (CDC) - United States
item SORVILLO, TERESA - Centers For Disease Control And Prevention (CDC) - United States
item COLEMAN-MCCRAY, JOANN - Centers For Disease Control And Prevention (CDC) - United States
item MARTIN, MARIA LAURA - National Institute Of Human Viral Disease
item BRIGNONE, JULIA - Centers For Disease Control And Prevention (CDC) - United States
item MONTGOMERY, JOEL - Centers For Disease Control And Prevention (CDC) - United States
item SPIROPOULOU, CHRISTINA - Centers For Disease Control And Prevention (CDC) - United States
item SPENGLER, JESSICA - Centers For Disease Control And Prevention (CDC) - United States

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2023
Publication Date: 11/8/2023
Citation: Davies, K.A., Welch, S.R., Sorvillo, T.E., Coleman-Mccray, J.D., Martin, M., Brignone, J.M., Montgomery, J.M., Spiropoulou, C.F., Spengler, J.R. 2023. Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses. Scientific Reports. 13. https://doi.org/10.1038/s41598-023-45740-w.
DOI: https://doi.org/10.1038/s41598-023-45740-w

Interpretive Summary: The research in this paper was performed to aid in the development of testing protocols for the detection of viruses that are encoded by RNA. When evaluating test results it is essential that a test be well defined and that there are controls to indicated that the test was performed correctly or the results were valid. This paper identified and evaluate potential positive controls that can be used when trying to measure the presence of viruses in different tissues.

Technical Abstract: Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean-Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.