Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue.
Abstract: West Nile virus (WNV), a small, positive sense, single stranded RNA virus continues to encroach into new locales with emergence of new viral variants. Neurological disease in the equine can be moderate to severe in the face of low to undetectable virus loads. Physical methods of virus enrichment may increase sensitivity of virus detection and enhance analysis of viral diversity, especially for deep sequencing studies. However, the use of these techniques is limited mainly to non-neural tissues. We investigated the hypothesis that elimination of equine brain RNA enhances viral detection without limiting viral variation. Eight different WNV viral RNA enrichment and host RNA separation methods were evaluated to determine if elimination of host RNA enhanced detection of WNV and increase the repertoire of virus variants for sequencing. Archived brain tissue from 21 different horses was inoculated with WNV, homogenized, before enrichment and separation. The protocols utilized combinations of low-speed centrifugation, syringe filtration, and nuclease treatment. Viral and host RNA were analyzed using real-time PCR targeting the WNV Envelope (E) protein and equine G3PDH to determine relative sensitivity for WNV and host depletion, respectively. To determine the effect of these methods on viral variation, deep sequencing of the E protein was performed. Our results demonstrate that additional separation and enrichment methods resulted in loss of virus in the face of host RNA depletion. DNA sequencing showed no significant difference in total sequence variation between the RNA enrichment protocols. For equine brain infected with WNV, direct RNA extraction followed by host RNA depletion was most suitable. This study highlights the importance of evaluating viral enrichment and separation methods according to tissue type before embarking on studies where quantification of virus and viral variants is essential to the outcome of the study.
Publication Date: 2018-12-18 PubMed ID: 30619900PubMed Central: PMC6305279DOI: 10.3389/fvets.2018.00318Google Scholar: Lookup
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- Journal Article
Summary
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The article investigates whether elimination of horse brain RNA would improve the detection of West Nile Virus (WNV) without affecting the structure of the virus. Eight different methods of virus enrichment and host RNA separation were tested and compared. The study concluded that while RNA enrichment techniques would lead to a loss of virus in the face of host RNA depletion, the total sequence variation remained unchanged among the different methods. The research findings underscore the necessity of testing out diverse viral extraction and enrichment processes according to the tissue type before undertaking studies in which accurate counting and identification of virus and viral variants are critical.
Methodology
- The study involved the use of archived horse brain tissue which was infected with the West Nile Virus (WNV) and then underwent homogenization prior to the process of enrichment and separation.
- The researchers used eight distinct methods of viral RNA enrichment and host RNA separation. These protocols combined different techniques such as low-speed centrifugation, syringe filtration and nuclease treatment.
- Real-time PCR targeting the WNV Envelope (E) protein and the equine G3PDH were used to analyze the virus and host RNA. This helped determine the relative sensitivity for WNV and host depletion.
- Finally, to evaluate the impact of the enrichment and separation methods on the virus variation, deep sequencing of the E protein was performed.
Key Findings
- Application of additional separation and enrichment techniques led to a loss of the virus while the host RNA was being depleted.
- Despite the loss of the virus, DNA sequencing revealed no significant changes in the total sequence variation between the different RNA enrichment methodologies employed.
- The researchers recognized that for horse brain infections with WNV, direct RNA extraction followed by host RNA depletion proved to be the most effective approach.
Implications
- The research highlights the need for careful consideration of the viral enrichment and separation methodologies used in studies, especially those where an accurate quantification of the virus and its variants are crucial to obtaining meaningful results.
- It emphasizes that such methodologies should be selected based on the tissue type being used in the study, to minimize possible errors and elicit more accurate and valid results.
Cite This Article
APA
Prakoso D, Dark MJ, Barbet AF, Salemi M, Barr KL, Liu JJ, Wenzlow N, Waltzek TB, Long MT.
(2018).
Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue.
Front Vet Sci, 5, 318.
https://doi.org/10.3389/fvets.2018.00318 Publication
Researcher Affiliations
- Department of Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
- Department of Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
- Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
- Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, United States.
- Department of Biology, Baylor University, Waco, TX, United States.
- Department of Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
- Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, QC, Canada.
- Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
- Department of Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
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Citations
This article has been cited 1 times.- Cerutti F, Giorda F, Grattarola C, Mignone W, Beltramo C, Keck N, Lorusso A, Di Francesco G, Di Renzo L, Di Guardo G, Goria M, Masoero L, Acutis PL, Casalone C, Peletto S. Specific capture and whole-genome phylogeography of Dolphin morbillivirus.. Sci Rep 2020 Nov 30;10(1):20831.
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