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.2018 Nov 23;18(Suppl 1):170.
doi: 10.1186/s12866-018-1298-1.

RNA interference-based antiviral immune response against the salivary gland hypertrophy virus in Glossina pallidipes

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Free PMC article

RNA interference-based antiviral immune response against the salivary gland hypertrophy virus in Glossina pallidipes

Irene K Meki et al. BMC Microbiol..
Free PMC article

Abstract

Background: Glossina 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).

Results: We 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.

Conclusion: The 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.

Keywords: Covert infections; Glossinidae; GpSGHV; RNAi; Sterile insect technique; Symptomatic and asymptomatic infection; Tsetse.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Phylogenetic and domain analysis of Argonaute proteins.a Maximum-likelihood based phylogenetic analysis (1000 bootstrap replicates) of Argonaute amino acid sequences of six tsetse species based on full length alignment withD. melanogaster as an outgroup.b Domain architecture of Argonaute proteins. The numbers on the domains are the scores produced by the ScanProsite search compared to the PROSITE protein domain database. All the tsetse AGO1, AGO2, and AGO3 proteins show similarity in the domain architecture to their orthologs inD. melanogaster (Dmel-AGO1, Dmel-AGO2 and Dmel-AGO3), respectively. Abbreviations; AGO (Argonaute), Gp (G. pallidipes), Gmm (G. m. morsitans), Gff (G. f. fuscipes), Gbr (G. brevipalpis), Gpp (G. p. palpalis), Gaus (G. austeni) and Dmel (D. melanogaster)
Fig. 2
Fig. 2
Phylogenetic and domain analysis of Dicer and Drosha proteins.a Maximum-likelihood based phylogenetic analysis (1000 bootstrap replicates) of Dicer and Drosha amino acid sequences of six tsetse species based on full length alignment withD. melanogaster orthologs as outgroup.b Domain architecture of Dicer and Drosha proteins. Some of the tsetse species had either DCR1 or DCR2 proteins, but Drosha was found in all the species. The numbers on the domains are the scores produced by the ScanProsite search compared to the PROSITE protein domain database. All DCR1, DCR2 and Drosha proteins show similarity in the domain architectures to Dmel-DCR1, Dmel-DCR2 and Dmel-Drosha, respectively. Abbreviations; DCR (Dicer), Gp (G. pallidipes), Gmm (G. m. morsitans), Gff (G. f. fuscipes), Gbr (G. brevipalpis), Gpp (G. p. palpalis), Gaus (G. austeni) and Dmel (D. melanogaster)
Fig. 3
Fig. 3
Relative expression of GpSGHVodv-e66 and RNAi pathway genes post GpSGHV (black line) or PBS (grey dotted line) injection inG. pallidipes flies.a) GpSGHVodv-e66;bAGO1;cAGO2;dAGO3;eDCR2; andfDrosha. Gene expression was quantified by RT-qPCR of the RNA extracted from whole fly bodies. Gene expression values were normalized toβ–tubulin and transformed by the Box-Cox process. The expression levels ofAGO1,AGO2 andDrosha were transformed using the lambda (λ) values (Expressionλ - 1)/λ), while virusodv-e66,AGO3 andDCR2 expressions were log transformed (log(Expression). The results from PBS and virus injection marked with the same lower-case letter do not differ at the 0.05 level
Fig. 4
Fig. 4
Comparative expression analysis of GpSGHVodv-e66 and RNAi pathway genes in asymptomatically and symptomatically infectedG. pallidipes flies.a Virusodv-e66 expression andb RNAi genes expression. Gene expression was quantified by RT-qPCR of the RNA extracted from whole fly bodies. Gene expression values were normalized toβ–tubulin and transformed by the Box-Cox process (log(Expression). The RNAi pathways in which the genes may be involved are also shown. Open boxes = asymptomatic infected; grey boxes = symptomatic infected. Asterisks indicate the statistical significance: ***P < 0.001, **P < 0.01, *P < 0.05
Fig. 5
Fig. 5
Validation of knockdown ofAGO2 andtsetse EP (control) genes inG. pallidipes. RT-qPCR expression analysis of:a)AGO2; andb)tsetse EP post PBS/virus injection, followingAGO2 andtsetse EP dsRNAs injection, respectively, compared to water injected flies (negative controls). Gene expression values were normalized toβ–tubulin and transformed by the Box-Cox process (Expressionλ - 1)/λ). Regression lines marked with the same lower-case letter do not differ at the 0.05 level
Fig. 6
Fig. 6
Effect ofAGO2 silencing on selected GpSGHV transcript levels inG. pallidipes, followingAGO2 knockdown. RT-qPCR expression analysis of (a) GpSGHVodv-e66 gene, (b) GpSGHVdnapol gene, (c) GpSGHV tegument gene and (d) GpSGHV capsid gene post virus injection, followingAGO2 andtsetse EP (control) dsRNAs injection. Gene expression values were normalized toβ–tubulin gene and transformed by the Box-Cox process (log(Expression)). Regression lines marked with the same lower-case letter do not differ at the 0.05 level
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References

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