Abstract
BackgroundGlossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded dsDNA virus that specifically infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae). GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can ultimately lead to a colony collapse. It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host’s mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. Here, we investigated the involvement of RNAi during GpSGHV infections by comparing the expression of three key RNAi machinery genes, Dicer (DCR), Argonaute (AGO) and Drosha, in artificially virus injected, asymptomatic and symptomatic infected G. pallidipes flies compared to PBS injected (controls) individuals. We further assessed the impact of AGO2 knockdown on virus infection by RT-qPCR quantification of four selected GpSGHV genes, i.e. odv-e66, dnapol, maltodextrin glycosyltransferase (a tegument gene) and SGHV091 (a capsid gene).ResultsWe show that in response to hemocoelic injections of GpSGHV into G. pallidipes flies, increased virus replication was accompanied by significant upregulation of the expression of three RNAi key genes; AGO1, AGO2 and DCR2, and a moderate increase in the expression of Drosha post injection compared to the PBS-injected controls. Furthermore, compared to asymptomatically infected individuals, symptomatic flies showed significant downregulation of AGO1, AGO2 and Drosha, but a moderate increase in the expression of DCR2. Compared to the controls, knockdown of AGO2 did not have a significant impact on virus infection in the flies as evidenced by unaltered transcript levels of the selected GpSGHV genes.ConclusionThe upregulation of the expression of the RNAi genes implicate involvement of this machinery in controlling GpSGHV infections and the establishment of symptomatic GpSGHV infections in Glossina. These findings provide a strategic foundation to understand GpSGHV infections and to control latent (asymptomatic) infections in Glossina spp. and thereby control SGHVs in insect production facilities.
Highlights
Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded doublestranded DNA (dsDNA) virus that infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae)
The Argonaute family in Glossina species The analyses of the genomes of G. pallidipes, G. m. morsitans, G. f. fuscipes, G. p. palpalis, G. austeni and G. brevipalpis resulted in the identification of AGO 1, 2 and 3 in all these species (See Table 1), key components of the RNA induced silencing complex (RISC); AGOs activate and cleave target messenger RNA (mRNA) within the RISC complex [36]
The three AGO proteins segregated into distinct clusters with their orthologs in D. melanogaster, which corresponded to the short interfering RNA (siRNA), microRNA and Piwi-interacting RNA (piRNA) pathways of the RNA interference (RNAi) machinery (Fig. 1a)
Summary
Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded dsDNA virus that infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae). GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can lead to a colony collapse It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host’s mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. In the mass rearing of G. pallidipes, SGH epizootics reduce fly survival and productivity, and have caused the collapse of three colonies; two in the Insect Pest Control Laboratory (IPCL) in Seibersdorf, Austria (in 1987 and 2001), and one in the mass rearing facility in Kality, Ethiopia (in 2012) [6] These GpSGHV-induced effects have significantly compromised the implementation of the sterile insect technique (SIT), a component of area-wide integrated pest management (AW-IPM) strategies designed for the eradication of G. pallidipes from the Southern Rift Valley of Ethiopia [7]. The viral infection is kept under control (asymptomatic state), but is not completely eliminated from the fly
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