Deletion of the NSm virulence gene of Rift Valley fever virus inhibits virus replication in and dissemination from the midgut of Aedes aegypti mosquitoes.

Barry R. Miller,Brad J. Biggerstaff,Rebekah C. Kading,Stuart T. Nichol,Mary B. Crabtree,Michael J. Turell,Brian H. Bird,Bobbie Rae Erickson,Kalanthe Horiuchi

doi:10.1371/journal.pntd.0002670

Barry R. Miller, Brad J. Biggerstaff + Show 7 more

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https://doi.org/10.1371/journal.pntd.0002670

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Abstract

BackgroundPreviously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection.Methodology and Principal Findings Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci.Conclusions/SignificanceDeletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.

Highlights

Rift Valley fever virus (RVFV) is a zoonotic, mosquito-borne virus endemic to Africa.Human illness is typically febrile, but 1–2% of cases develop more severe disease including hepatitis, encephalitis, retinitis, vision loss, jaundice, severe anemia, neurologic manifestations, renal failure, and hemorrhagic fever [1,2,3]
To maximize infectivity to mosquitoes, freshly-harvested rRVFwt and rRVF-ÄNSm virus strains were used in the infectious blood meal
We found that midgut infections in mosquitoes exposed to rRVF-wt were extensive, whereas midgut infections in mosquitoes infected with rRVF-DNSm were confined to only one or a few small foci

Summary

Introduction

Rift Valley fever virus (RVFV) (family Bunyaviridae, genusPhlebovirus) is a zoonotic, mosquito-borne virus endemic to Africa.Human illness is typically febrile, but 1–2% of cases develop more severe disease including hepatitis, encephalitis, retinitis, vision loss, jaundice, severe anemia, neurologic manifestations, renal failure, and hemorrhagic fever [1,2,3]. A hallmark of RVFV outbreaks are ‘‘abortion storms’’ among sheep and cattle, with devastating mortality rates in newborn and young animals [4,5]. RVFV is registered as a Category A overlap select agent with both the U.S Department of Agriculture Animal and Plant Health Inspection Service and the Centers for Disease Control and Prevention due to its biothreat potential and ability to cause significant economic losses to the livestock industry as well as substantial human morbidity and mortality [6,7]. We investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-DNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection

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