Student Major/Year in School
Biology, Pre-med, third year
Faculty Mentor Information
A. Sally Davis, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine
Abstract
Middle Eastern respiratory syndrome (MERS), caused by MERS coronavirus (MERS-CoV) first appeared in Saudi Arabia in 2012. Although there have been less than 2,400 cases reported, the case fatality rate is 35%. Dromedary camels are the known host animal for the virus. However, they are difficult to manage experimental animals. In a recent study, alpacas proved to be a suitable experimental animal to further characterize MERS-CoV infection. Our work is part of a wider project focused on studying the efficacy of MERS-CoV antigen and RNA labeling in infected alpaca tissues, when these tissues are preserved in novel modular alcohol fixatives compared to 10% neutral buffered formalin (NBF). These new fixatives have superior molecular properties, equivalent histomorpohology, and like NBF also inactivate the virus. In order to examine these new fixatives further, we first validated immunohistochemistry (IHC) for MERS-CoV antigen in formalin-fixed, paraffin-embedded (FFPE) alpaca tissues to serve as an experimental control. The tissues were subsampled, processed, paraffin-embedded and blocks sectioned for immunohistochemistry. Similarly prepared slides of alpaca trachea from a US animal not possibly infected with MERS, provided by the KS Veterinary Diagnostic Laboratory, served as an uninfected control. Multiple IHC detection systems were tested. The polymer-based horseradish peroxidase technique provided the best result, least background and a reliable and biologically appropriate signal. Uninfected alpaca trachea and reagent controls on infected tissues, which included use of isotype antibody, had no background. Labeling in the nasal turbinate epithelium was typically stronger and more abundant than in the tracheal epithelium, which correlates with expected virus distribution in the MERS alpaca model. Future directions include more detailed analysis of IHC for all the FFPE alpaca tissues, optimization of IHC for matched tissues preserved in the novel alcohol fixatives as well as correlation of these IHC results with MERS-CoV in situ hybridization, which labels viral RNA instead of viral antigen.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Recommended Citation
Carlos, Elena (2019). "Comparison of Immunohistochemistry Methods for Visualization of Middle Eastern Respiratory Viral Antigen," Kansas State University Undergraduate Research Conference. https://newprairiepress.org/ksuugradresearch/2019/posters/53
Comparison of Immunohistochemistry Methods for Visualization of Middle Eastern Respiratory Viral Antigen
Middle Eastern respiratory syndrome (MERS), caused by MERS coronavirus (MERS-CoV) first appeared in Saudi Arabia in 2012. Although there have been less than 2,400 cases reported, the case fatality rate is 35%. Dromedary camels are the known host animal for the virus. However, they are difficult to manage experimental animals. In a recent study, alpacas proved to be a suitable experimental animal to further characterize MERS-CoV infection. Our work is part of a wider project focused on studying the efficacy of MERS-CoV antigen and RNA labeling in infected alpaca tissues, when these tissues are preserved in novel modular alcohol fixatives compared to 10% neutral buffered formalin (NBF). These new fixatives have superior molecular properties, equivalent histomorpohology, and like NBF also inactivate the virus. In order to examine these new fixatives further, we first validated immunohistochemistry (IHC) for MERS-CoV antigen in formalin-fixed, paraffin-embedded (FFPE) alpaca tissues to serve as an experimental control. The tissues were subsampled, processed, paraffin-embedded and blocks sectioned for immunohistochemistry. Similarly prepared slides of alpaca trachea from a US animal not possibly infected with MERS, provided by the KS Veterinary Diagnostic Laboratory, served as an uninfected control. Multiple IHC detection systems were tested. The polymer-based horseradish peroxidase technique provided the best result, least background and a reliable and biologically appropriate signal. Uninfected alpaca trachea and reagent controls on infected tissues, which included use of isotype antibody, had no background. Labeling in the nasal turbinate epithelium was typically stronger and more abundant than in the tracheal epithelium, which correlates with expected virus distribution in the MERS alpaca model. Future directions include more detailed analysis of IHC for all the FFPE alpaca tissues, optimization of IHC for matched tissues preserved in the novel alcohol fixatives as well as correlation of these IHC results with MERS-CoV in situ hybridization, which labels viral RNA instead of viral antigen.
