Bioluminescent Imaging and Histopathologic Characterization of WEEV Neuroinvasion in Outbred CD-1 Mice

Aaron T Phillips,Ken E Olson,Charles B Stauft,Donald L Jarvis,Tawfik A Aboellail,Ann M Powers,Ann M Toth,Patricia V Aguilar

doi:10.1371/journal.pone.0053462

Aaron T Phillips, Ken E Olson + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0053462

Copy DOI

Abstract

Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection. Here in vivo and ex vivo bioluminescence (BLM) imaging was applied on mice intranasally infected with a recombinant McM virus expressing firefly luciferase (FLUC) to track viral neuroinvasion by FLUC detection and determine any correlation between BLM and viral titer. Immunological markers of disease (MCP-1 and IP-10) were measured and compared to wild type virus infection. Histopathology was guided by corresponding BLM images, and showed that neuroinvasion occurred primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected with subsequent spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. The recombinant virus WEEV.McM.FLUC had attenuated replication kinetics and induced a weaker immunological response than WEEV.McM but produced comparable pathologies. Immunohistochemistry staining for FLUC and WEEV antigen showed that transgene expression was present in all areas of the CNS where virus was observed. BLM provides a quantifiable measure of alphaviral neural disease progression and a method for evaluating antiviral strategies.

Highlights

Of the 29 mosquito-borne viral species within the Alphavirus genus (Togaviridae), at least 16 are known to cause disease in humans and animals [1,2,3,4]
A 0% survival rate was observed for both western equine encephalitis virus (WEEV).McM and WEEV.McM.Firefly luciferase (FLUC) in animals (n = 10) inoculated by the intranasal route and a comparison of mouse survival showed no significant difference between the two viruses (Figure 1D) (P value = 0.4795)
WEEV.McM virus replicated to 100- fold PFU/ mL higher titer than WEEV.McM.FLUC within the first 24 hpi

Summary

Introduction

Of the 29 mosquito-borne viral species within the Alphavirus genus (Togaviridae), at least 16 are known to cause disease in humans and animals [1,2,3,4]. WEEV was first isolated from an outbreak of equine encephalitis in the San Joaquin Valley of California that affected almost 6,000 horses and was associated with an equine mortality rate of 50% [6]. According to the USDA, epizootics have been reported in horses (Canada 1975), turkeys (California 1993– 1994; Nebraska 1957), and emus (Texas and Oklahoma 1992). These findings highlight the potential for a WEEV epidemic outbreak in humans. Human patients may present clinically with symptoms ranging from an acute febrile illness to fulminant encephalitis. Among the high mortality phenotypes, McM induces rapid and lethal encephalitic disease in a mouse infection model and is the basis of the data reported here

Methods

Results

Conclusion