Detection of Rift Valley Fever Virus RNA in Formalin-Fixed Mosquitoes by In Situ Hybridization (RNAscope®).

Sarah Lumley,Anthony R Fooks,Nicholas Johnson,Laura Hunter,Daniel L Horton,Kirsty Emery,Roger Hewson

doi:10.3390/v13061079

Sarah Lumley, Anthony R Fooks + Show 5 more

Open Access

https://doi.org/10.3390/v13061079

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Abstract

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne haemorrhagic disease that emerges to produce rapid large-scale outbreaks in livestock within sub-Saharan Africa. A range of mosquito species in Africa have been shown to transmit RVFV, and recent studies have assessed whether temperate mosquito species are also capable of transmission. In order to support vector competence studies, the ability to visualize virus localization in mosquito cells and tissue would enhance the understanding of the infection process within the mosquito body. Here, the application of in situ hybridization utilizing RNAscope® to detect RVFV infection within the mosquito species, Culex pipiens, derived from the United Kingdom was demonstrated. Extensive RVFV replication was detected in many tissues of the mosquito with the notable exception of the interior of ovarian follicles.

Highlights

Rift Valley fever virus is transmitted to and between mammals by mosquitoes, human infections usually result from close contact with contaminated carcasses
Each mosquito was fixed in formalin, and preliminary sections were stained at 25 °C
Each mosquito was fixed in formalin, and preliminary sections were stained with haematoxylin and eosin (Figure 1) to identify key structures

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rift Valley fever virus is transmitted to and between mammals by mosquitoes, human infections usually result from close contact with contaminated carcasses. The ability to detect the virus in the context of organ structures, using immunohistochemistry, provides further information on the ability of arthropod-borne viruses to infect particular mosquito species [7,8]. A recent report has developed RNAscope® for detection of RVFV within mammalian liver tissue [10], a key tissue for diagnosis in livestock, using probes that target the virus polymerase gene on the L segment. We have adapted a similar approach, but using probes designed against the virus nucleoprotein gene on the S segment, to investigate the presence and distribution of RVFV RNA within an infected mosquito. Staining with RVFV probes demonstrated extensive RVFV RNA labelling in key structures such as the basal layer of the mosquito midgut and the proventriculus, suggesting points of entry for the virus following consumption of a bloodmeal

Materials and Methods

In Situ Hybridization

Results and Discussion

Conclusions