Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 3;2(3):e00090-17.
doi: 10.1128/mSphere.00090-17. eCollection 2017 May-Jun.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems

Isabelle Dietrich  1 , Stephanie Jansen  2 , Gamou Fall  3 , Stephan Lorenzen  2 , Martin Rudolf  2 , Katrin Huber  2   4 , Anna Heitmann  2 , Sabine Schicht  5 , El Hadji Ndiaye  6 , Mick Watson  7 , Ilaria Castelli  8 , Benjamin Brennan  1 , Richard M Elliott  1 , Mawlouth Diallo  6 , Amadou A Sall  3 , Anna-Bella Failloux  8 , Esther Schnettler  1   2 , Alain Kohl  1 , Stefanie C Becker  2   5
Affiliations

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems

Isabelle Dietrich et al. mSphere. .

Abstract

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

Keywords: Drosophila melanogaster; RNA interference; Rift Valley fever virus; antiviral immunity; mosquito.

PubMed Disclaimer

Figures

FIG 1
FIG 1
RVFV MP12-infected Aag2 and S2 cells produce virus-specific small RNAs. (A and B) Small-RNA reads matching the RVFV MP12 genomic/antigenomic sequences were plotted against the corresponding read lengths (in nucleotides [nt]) for (A) Aag2 cells infected for 24 h and (B) D. melanogaster S2 cells infected for 96 h prior to small-RNA extraction. (C and D) Alignment of the 21-nt small-RNA reads to the RVFV MP12 sequences of the three RNA segments. The x axis represents the position of small-RNA reads in reference to the antigenome RNA sequence (5′-to-3′ orientation) or the genome orientation (3′-to-5′ orientation). The y axis represents the number of reads for each position, with reads matching the antigenome shown on the positive y axis and reads matching the genome shown on the negative y axis. (C) Aag2 cells infected for 24 h. (D) D. melanogaster S2 cells infected for 96 h prior to small-RNA extraction.
FIG 2
FIG 2
RVFV-specific piRNA-like small RNAs are produced in infected Aedes aegypti Aag2 cells. (A) Representation of piRNA sequence logos within the 28-nt reads. All reads matching the RVFV MP12 genomic and antigenomic sequences were analyzed for their base frequencies at each position. The x axis shows the nucleotide position. The y axis represents the frequency of each nucleotide at the corresponding position. (B) The panel shows the probability of overlap at the 5′ end of the opposite viral small RNA for the 28-nt reads. The x axis indicates the number of nucleotides overlapping between the antigenomic and genomic sequences; the y axis shows the mean fractions of reads overlapping. (C) Alignment of the 28-nt small-RNA reads to the RVFV MP12 sequences of the three viral RNA segments. The x axis represents the position of small-RNA reads in reference to the antigenome RNA sequence (5′-to-3′ orientation) or to the genome (3′-to-5′ orientation). The y axis represents the number of reads for each position, with reads matching the antigenome shown on the positive y axis and reads matching the genome shown on the negative y axis.
FIG 3
FIG 3
RVFV-derived small RNAs are present in three different vector mosquito species. The small-RNA fraction from RVFV MP12-infected A. aegypti, A. vexans, and C. quinquefasciatus whole mosquito bodies was isolated 14 days p.i. (A. aegypti and C. quinquefasciatus) or 15 days p.i. (A. vexans) and sequenced (A to C). The graphs represent the small-RNA reads mapping to the RVFV MP12 genomic and antigenomic sequences. Read numbers per length were plotted for A. aegypti (A), A. vexans (B), and C. quinquefasciatus (C) against the corresponding read length (in nucleotides) on a linear scale. (D to F) Alignment of the 21-nt small-RNA reads mapping to the RVFV MP12 sequences of the three RNA segments for all mosquito species as described for RVFV-derived small-RNA reads in Aag2 cells (Fig. 1C). The alignments for A. aegypti (D), A. vexans (E), and C. quinquefasciatus (F) are shown.
FIG 4
FIG 4
RVFV-specific piRNA-like small RNAs are produced in infected mosquitoes. (A to C) Representation of piRNA sequence logos within 28-nt reads. All reads mapping to the RVFV MP12 genomic and antigenomic sequences were analyzed for their base frequencies at each position for RVFV-derived small-RNA reads as previously described in Aag2 cells (Fig. 2A). A. aegypti data are presented in panel A, A. vexans data are shown in panel B, and C. quinquefasciatus data are shown in panel C. (D to F) The panels show the probability of overlap at the 5′ end of the opposite viral small RNA for the 28-nt reads. The x axis indicates the number of nucleotides overlapping between the antigenomic and genomic sequences; the y axis shows the mean fractions of reads overlapping. A. aegypti data are presented in panel D, A. vexans data are shown in panel E, and C. quinquefasciatus data are shown in panel F. (G to I) Alignment of the 28-nt small-RNA reads to RVFV MP12 RNA segments as previously described for RVFV-derived small-RNA reads in Aag2 cells (Fig. 2C). The alignment of A. aegypti is presented in panel G, that of A. vexans is shown in panel H, and that of C. quinquefasciatus is shown in in panel I.
FIG 5
FIG 5
RVFV-derived small RNAs display antiviral properties. (A and B) The accessibility of RVFV mRNAs to RVFV-specific small RNAs and their antiviral properties were investigated using luciferase reporter-based sensor constructs (A) and RVFV-targeting dsRNAs (B), respectively. (A) Aag2 cells were either infected with RVFV MP12 (white bars) or left uninfected (black bars). At 24 h p.i., cells were transfected with small-RNA sensors (pIZ containing a nanoluciferase [NLuc] ORF fused to RVFV-specific/control sequences) and a transfection control (pIZ-FLuc). RVFV-specific sequences consisted of fragments amplified from the L open-reading frame (ORF; 515 nt), the M ORF (529 nt), the N ORF (498 nt), or the 5′ or 3′ halves of the NSs ORF (NSs5′, 416 nt; NSs3′, 329 nt). Primer binding sites for the respective fragments are indicated in Table S3. NLuc/FLuc ratios in transfected infected cells were normalized against the respective transfected uninfected cells. The graph presents means and standard errors of results from three independent experiments, each performed in triplicate. Statistical analysis was performed using the t test. *, P < 0.05. (B) dsRNAs targeting the RVFV rMP12delNSs:hRen L, M, and S segments (dsL, dsM, dsN, dshRen) or a control dsRNA targeting eGFP (dsCon) were transfected into Aag2 cells, followed by virus infection. Viral replication was measured by luciferase assay, and dsL, dsM, dsN, and dshRen transfected samples were normalized against the control samples. The graph presents means and standard errors of results from three independent experiments, each performed in triplicate. Statistical analysis was performed using the t test. *, P < 0.05.
FIG 6
FIG 6
RNAi pathways limit replication of RVFV in A. aegypti and D. melanogaster cells. (A and B) Impact of Ago2 knockdown on replication of RVFV rMP12delNSs:hRen in Aag2 cells (A) or D. melanogaster S2 cells (B). Knockdown of Ago2 was induced by transfection of dsRNA targeting the corresponding sequence (A. aegypti dsAgo2 in panel A or D. melanogaster dsDmAgo2 in panel B). Cells transfected with dsRNA targeting eGFP (dsCon) were used as controls. Viral replication was measured in dsAgo2-treated Aag2 and S2 cells by luciferase assay; values were normalized against the respective dsCon-treated cells. (A) The graph presents means and standard errors of results from five independent experiments performed in triplicate. (B) The graph presents means and standard errors of results from four independent experiments, each performed in sextuplicate. Statistical analysis was performed using the t test. *, P < 0.05. (C) Impact of Piwi4, Piwi5, Piwi6, and Ago3 knockdown on replication of RVFV rMP12delNSs:hRen in Aag2 cells. Knockdown of Piwi4, Piwi5, Piwi6, and Ago3 was induced by transfection of dsRNA targeting the corresponding sequence, and dsRNA targeting eGFP (dsCon) was used as a control. Viral replication in dsPiwi4-, dsPiwi5-, dsPiwi6-, and dsAgo3-treated cells as measured by luciferase assay was normalized against that in control (dsCon-treated) cells. The panel represents mean values and standard errors of results from five independent experiments performed in triplicate. Statistical analysis was performed using the t test. *, P < 0.05.
FIG 7
FIG 7
RVFV does not interfere with the exogenous RNAi pathway in mosquito and D. melanogaster cells. (A and B) The effect of RVFV MP12 protein expression on the induction of exogenous RNAi was analyzed using a FLuc reporter system. (A) Aag2 cells were infected with RVFV MP12 or Culex Y virus (CYV) as a control or left uninfected and subsequently transfected with luciferase expression plasmids (pIZ-FLuc as the reporter and pAct-Renilla as an internal control) and dsRNA targeting either FLuc or eGFP control sequences. Values of specific luciferase activity reduction were first normalized against a transfection control, and additional specific FLuc dsRNA-transfected samples were normalized against dseGFP-transfected controls. Effects of virus infection on RNAi induction are presented as percent silencing compared to the noninfected cells. The graph presents means and standard errors of results from three independent experiments performed in triplicate. Statistical analysis was performed using the t test. *, P < 0.05. (B) S2 cells were transfected with luciferase expression plasmids (pMT-Fluc as the reporter and pMT-Rluc as an internal control) and dsRNA targeting either FLuc or eGFP as a control. Cells were then either infected with RVFV MP12 or left uninfected. dsRNA targeting either FLuc or eGFP as a control was added to the cell culture media. The expression of luciferase reporters was induced and quantified. Data were analyzed and presented as described for panel A. The graph presents means and standard errors of results from three independent experiments performed in sextuplicate. Statistical analysis was performed using the t test. *, P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Daubney R, Hudson JR, Garnham PC. 1931. Enzootic hepatitis or rift valley fever. An undescribed virus disease of sheep cattle and man from east africa. J Pathol 34:545–579. doi:10.1002/path.1700340418. - DOI
    1. Gerdes GH. 2004. Rift Valley fever. Rev Sci Tech 23:613–623. doi:10.20506/rst.23.2.1500. - DOI - PubMed
    1. Smithburn KC. 1949. Rift Valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine. Br J Exp Pathol 30:1–16. - PMC - PubMed
    1. Al-Hazmi M, Ayoola EA, Abdurahman M, Banzal S, Ashraf J, El-Bushra A, Hazmi A, Abdullah M, Abbo H, Elamin A, Al-Sammani el T, Gadour M, Menon C, Hamza M, Rahim I, Hafez M, Jambavalikar M, Arishi H, Aqeel A. 2003. Epidemic Rift Valley fever in Saudi Arabia: a clinical study of severe illness in humans. Clin Infect Dis 36:245–252. doi:10.1086/345671. - DOI - PubMed
    1. LaBeaud AD, Muiruri S, Sutherland LJ, Dahir S, Gildengorin G, Morrill J, Muchiri EM, Peters CJ, King CH. 2011. Postepidemic analysis of Rift Valley fever virus transmission in northeastern Kenya: a village cohort study. PLoS Negl Trop Dis 5:e1265. doi:10.1371/journal.pntd.0001265. - DOI - PMC - PubMed

LinkOut - more resources