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Front. Vet. Sci. | doi: 10.3389/fvets.2018.00318

Viral Enrichment Methods Affect the Detection but not Sequence Variation of West Nile virus in Equine Brain Tissue

 Dhani Prakoso1,  Michael J. Dark1, Anthony F. Barbet2, Marco Salemi3,  Kelli L. Barr1, Jun J. Liu1,  Nanny Wenzlow4, Thomas B. Waltzek2 and  Maureen T. Long1*
  • 1Department of Comparative Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, United States
  • 2Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, United States
  • 3Department of Pathology, College of Medicine, University of Florida, United States
  • 4Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Université de Montréal, Canada

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.

Keywords: West Nile virus, Arbovirus, Equine, RNA Extraction, Viral enrichment, Viral variation

Received: 04 Aug 2018; Accepted: 29 Nov 2018.

Edited by:

Lester J. Perez, Faculty of Medicine, Dalhousie University, Canada

Reviewed by:

Francisco Rivera-Benítez, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Mexico
Irit Davidson, Kimron Veterinary Institute, Israel  

Copyright: © 2018 Prakoso, Dark, Barbet, Salemi, Barr, Liu, Wenzlow, Waltzek and Long. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: DVM, PhD. Maureen T. Long, College of Veterinary Medicine, University of Florida, Department of Comparative Diagnostic and Population Medicine, Gainesville, 32608, Texas, United States,