Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue.

Dhani Prakoso,Nanny Wenzlow,Maureen T Long,Thomas B Waltzek,Anthony F Barbet,Michael J Dark,Marco Salemi,Junjie J Liu,Kelli L Barr

doi:10.3389/fvets.2018.00318

Dhani Prakoso, Nanny Wenzlow + Show 7 more

Open Access

https://doi.org/10.3389/fvets.2018.00318

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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.

Highlights

During 1999, West Nile virus (WNV) was introduced to North America resulting in more than 49,794 human infections with 4% mortality, 28,063 equine infections, and over 100,000 avian population infections [1, 2]
Low average RNA integrity number (RIN) numbers were measured from treatment groups TR, CTR, and CSNTR with average RINs of 2.3, 2.024, and 2.475, respectively
The treatment groups with RNA depletion with selective depletion of host ribosomal RNA had significantly lower RIN numbers compared to the groups which did not have the host RNA depletion treatment (p = 0.001)

Summary

Introduction

During 1999, West Nile virus (WNV) was introduced to North America resulting in more than 49,794 human infections with 4% mortality, 28,063 equine infections, and over 100,000 avian population infections [1, 2]. In the US during 2012, a new subtype of the US lineage 1 virus emerged with a substantial increase in reporting of human and horse disease [4, 5]. Diversification of WNV occurs through spontaneous mutation due to the poor proof-reading mechanisms inherent in positive-sense, single-stranded RNA viruses. This leads to the production of WNV variants are often associated with new tissues or cellular tropisms [7, 8] likely resulting in enhanced neurovirulence in humans and horses [9, 10]. Viral diversity of WNV increases following infection of a new type of host cell [11], understanding viral variation within equine tissues can enhance our understanding of WNV pathogenicity in this host [12]

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