Publications by authors named "Esther Schnettler"

52 Publications

An -Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections.

Viruses 2021 06 3;13(6). Epub 2021 Jun 3.

Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany.

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.
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http://dx.doi.org/10.3390/v13061066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227176PMC
June 2021

aBravo Is a Novel Antiviral Protein that Interacts with, but Acts Independently of, the Exogenous siRNA Pathway Effector Dicer 2.

Viruses 2020 07 11;12(7). Epub 2020 Jul 11.

MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK.

Mosquitoes, such as , can transmit arboviruses to humans. The exogenous short interfering RNA (exo-siRNA) pathway plays a major antiviral role in controlling virus infection in mosquito cells. The Dicer 2 (Dcr2) nuclease is a key effector protein in this pathway, which cleaves viral double-stranded RNA into virus-derived siRNAs that are further loaded onto an effector called Argonaute 2 (Ago2), which as part of the multiprotein RNA-induced silencing complex (RISC) targets and cleaves viral RNA. In order to better understand the effector protein Dcr2, proteomics experiments were conducted to identify interacting cellular partners. We identified several known interacting partners including Ago2, as well as two novel and previously uncharacterized proteins. The role of these two proteins was further investigated, and their interactions with Dcr2 verified by co-immunoprecipitation. Interestingly, despite their ability to interact with Ago2 and Piwi4, neither of these proteins was found to affect exo-siRNA silencing in a reporter assay. However, one of these proteins, Q0IFK9, subsequently called aBravo (edine oadly active ntiiral prtein), was found to mediate antiviral activity against positive strand RNA arboviruses. Intriguingly the presence of Dcr2 was not necessary for this effect, suggesting that this interacting antiviral effector may act as part of protein complexes with potentially separate antiviral activities.
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http://dx.doi.org/10.3390/v12070748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411624PMC
July 2020

Identification and RNAi Profile of a Novel Iflavirus Infecting Senegalese Mosquitoes.

Viruses 2020 04 14;12(4). Epub 2020 Apr 14.

Institute for Parasitology, University of Veterinary Medicine Hannover, 30559 Hannover, Germany.

The inland floodwater mosquito (Meigen, 1830) is a competent vector of numerous arthropod-borne viruses such as Rift Valley fever virus () and Zika virus (). spp. have widespread Afrotropical distribution and are common European cosmopolitan mosquitoes. We examined the virome of samples from Barkédji village, Senegal, with small RNA sequencing, bioinformatic analysis, and RT-PCR screening. We identified a novel 9494 nt iflavirus () designated here as Aedes vexans iflavirus (AvIFV). Annotation of the AvIFV genome reveals a 2782 amino acid polyprotein with iflavirus protein domain architecture and typical iflavirus 5' internal ribosomal entry site and 3' poly-A tail. Aedes vexans iflavirus is most closely related to a partial virus sequence from (a parasitoid wasp) with 56.77% pairwise amino acid identity. Analysis of AvIFV-derived small RNAs suggests that AvIFV is targeted by the exogenous RNA interference pathway but not the PIWI-interacting RNA response, as ~60% of AvIFV reads corresponded to 21 nt Dicer-2 virus-derived small RNAs and the 24-29 nt AvIFV read population did not exhibit a "ping-pong" signature. The RT-PCR screens of archival and current (circa 2011-2020) laboratory samples and wild-caught mosquitoes from Barkédji suggest that AvIFV is ubiquitous in these mosquitoes. Further, we screened wild-caught European samples from Germany, the United Kingdom, Italy, and Sweden, all of which tested negative for AvIFV RNA. This report provides insight into the diversity of commensal viruses and the host RNAi response towards iflaviruses.
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http://dx.doi.org/10.3390/v12040440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232509PMC
April 2020

The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and Expression.

mSphere 2020 04 8;5(2). Epub 2020 Apr 8.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland

Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus () both in -derived cells and Antiviral activity of p400 was also demonstrated against chikungunya virus () and Bunyamwera virus (e) but not Zika virus (). p400 was found to be expressed across mosquito tissues and regulated but not transcript levels in mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein's antiviral and RNA interference regulatory activities in Female mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito's antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that p400, whose ortholog Domino in is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in mosquitoes.
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http://dx.doi.org/10.1128/mSphere.00081-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142294PMC
April 2020

Tick-borne encephalitis virus inhibits rRNA synthesis and host protein production in human cells of neural origin.

PLoS Negl Trop Dis 2019 09 27;13(9):e0007745. Epub 2019 Sep 27.

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, České Budějovice, Czech Republic.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off.
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http://dx.doi.org/10.1371/journal.pntd.0007745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785130PMC
September 2019

Mosquito-Specific Viruses-Transmission and Interaction.

Viruses 2019 09 17;11(9). Epub 2019 Sep 17.

Molecular Entomology, Molecular Biology and Immunology Department, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany.

Mosquito-specific viruses (MSVs) are a subset of insect-specific viruses that are found to infect mosquitoes or mosquito derived cells. There has been an increase in discoveries of novel MSVs in recent years. This has expanded our understanding of viral diversity and evolution but has also sparked questions concerning the transmission of these viruses and interactions with their hosts and its microbiome. In fact, there is already evidence that MSVs interact with the immune system of their host. This is especially interesting, since mosquitoes can be infected with both MSVs and arthropod-borne (arbo) viruses of public health concern. In this review, we give an update on the different MSVs discovered so far and describe current data on their transmission and interaction with the mosquito immune system as well as the effect MSVs could have on an arboviruses-co-infection. Lastly, we discuss potential uses of these viruses, including vector and transmission control.
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http://dx.doi.org/10.3390/v11090873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784079PMC
September 2019

Metaviromics Reveals Unknown Viral Diversity in the Biting Midge .

Viruses 2019 09 17;11(9). Epub 2019 Sep 17.

MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, UK.

Biting midges ( species) are vectors of arboviruses and were responsible for the emergence and spread of (SBV) in Europe in 2011 and are likely to be involved in the emergence of other arboviruses in Europe. Improved surveillance and better understanding of risks require a better understanding of the circulating viral diversity in these biting insects. In this study, we expand the sequence space of RNA viruses by identifying a number of novel RNA viruses from (biting midge) using a meta-transcriptomic approach. A novel metaviromic pipeline called MetaViC was developed specifically to identify novel virus sequence signatures from high throughput sequencing (HTS) datasets in the absence of a known host genome. MetaViC is a protein centric pipeline that looks for specific protein signatures in the reads and contigs generated as part of the pipeline. Several novel viruses, including an alphanodavirus with both segments, a novel relative of the Hubei sobemo-like virus 49, two rhabdo-like viruses and a chuvirus, were identified in the Scottish midge samples. The newly identified viruses were found to be phylogenetically distinct to those previous known. These findings expand our current knowledge of viral diversity in arthropods and especially in these understudied disease vectors.
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http://dx.doi.org/10.3390/v11090865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784199PMC
September 2019

Antiviral RNA Interference Activity in Cells of the Predatory Mosquito, .

Viruses 2018 12 6;10(12). Epub 2018 Dec 6.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland G61 1QH, UK.

Arthropod vectors control the replication of arboviruses through their innate antiviral immune responses. In particular, the RNA interference (RNAi) pathways are of notable significance for the control of viral infections. Although much has been done to understand the role of RNAi in vector populations, little is known about its importance in non-vector mosquito species. In this study, we investigated the presence of an RNAi response in , which is a non-blood feeding species proposed as a biological control agent against pest mosquitoes. Using a derived cell line (TRA-171), we demonstrate that these mosquitoes possess a functional RNAi response that is active against a mosquito-borne alphavirus, Semliki Forest virus. As observed in vector mosquito species, small RNAs are produced that target viral sequences. The size and characteristics of these small RNAs indicate that both the siRNA and piRNA pathways are induced in response to infection. Taken together, this data suggests that are able to control viral infections in a similar way to natural arbovirus vector mosquito species. Understanding their ability to manage arboviral infections will be advantageous when assessing these and similar species as biological control agents.
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http://dx.doi.org/10.3390/v10120694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316411PMC
December 2018

RNAi-mediated antiviral immunity in insects and their possible application.

Curr Opin Virol 2018 10 12;32:108-114. Epub 2018 Nov 12.

Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany; German Centre for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Hamburg 20359, Germany. Electronic address:

Antiviral RNA interference (RNAi) in insects is known for some time. Previously, data from the model organism Drosophila was lightly transferred to other insects. However, since more research is performed on other insects, both similarities and important differences among the RNAi pathways of insects are revealed. The piRNA pathway, for example, is suggested to have antiviral functions in mosquitoes, which is unknown for other insects. Moreover, production of viral cDNA from RNA viruses during infection and their possible incorporation into the genome hint towards potential inheritable immunity in mosquitoes. Increased knowledge of antiviral RNAi pathways has initiated efforts to engineer insects resistant to viral infections. New advances in genome editing will further stimulate this process.
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http://dx.doi.org/10.1016/j.coviro.2018.10.004DOI Listing
October 2018

Detection, infection dynamics and small RNA response against Culex Y virus in mosquito-derived cells.

J Gen Virol 2018 12 5;99(12):1739-1745. Epub 2018 Nov 5.

4​MRC - University of Glasgow Centre of Virus Research, Glasgow, UK.

Many insect cell lines are persistently infected with insect-specific viruses (ISV) often unrecognized by the scientific community. Considering recent findings showing the possibility of interference between arbovirus and ISV infections, it is important to pay attention to ISV-infected cell lines. One example is the Entomobirnavirus, Culex Y virus (CYV). Here we describe the detection of CYV using a combination of small RNA sequencing, electron microscopy and PCR in mosquito cell lines Aag2, U4.4 and C7-10. We found CYV-specific small RNAs in all three cell lines. Interestingly, the magnitude of the detected viral RNA genome is variable among cell passages and leads to irregular detection via electron microscopy. Gaining insights into the presence of persistent ISV infection in commonly used mosquito cells and their interactions with the host immune system is beneficial for evaluating the outcome of co-infections with arboviruses of public health concern.
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http://dx.doi.org/10.1099/jgv.0.001173DOI Listing
December 2018

The antiviral piRNA response in mosquitoes?

J Gen Virol 2018 12 29;99(12):1551-1562. Epub 2018 Oct 29.

2​Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.

There are several RNA interference (RNAi) pathways in insects. The small interfering RNA pathway is considered to be the main antiviral mechanism of the innate immune system; however, virus-specific P-element-induced Wimpy testis gene (PIWI)-interacting RNAs (vpiRNAs) have also been described, especially in mosquitoes. Understanding the antiviral potential of the RNAi pathways is important, given that many human and animal pathogens are transmitted by mosquitoes, such as Zika virus, dengue virus and chikungunya virus. In recent years, significant progress has been made to characterize the piRNA pathway in mosquitoes (including the possible antiviral activity) and to determine the differences between mosquitoes and the model organism Drosophila melanogaster. The new findings, especially regarding vpiRNA in mosquitoes, as well as important questions that need to be tackled in the future, are discussed in this review.
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http://dx.doi.org/10.1099/jgv.0.001157DOI Listing
December 2018

Spindle-E Acts Antivirally Against Alphaviruses in Mosquito Cells.

Viruses 2018 02 18;10(2). Epub 2018 Feb 18.

Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, Hamburg 20359, Germany.

Mosquitoes transmit several human- and animal-pathogenic alphaviruses ( family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in -derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in , resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes.
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http://dx.doi.org/10.3390/v10020088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850395PMC
February 2018

Characterization of the Zika virus induced small RNA response in Aedes aegypti cells.

PLoS Negl Trop Dis 2017 Oct 17;11(10):e0006010. Epub 2017 Oct 17.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

RNA interference (RNAi) controls arbovirus infections in mosquitoes. Two different RNAi pathways are involved in antiviral responses: the PIWI-interacting RNA (piRNA) and exogenous short interfering RNA (exo-siRNA) pathways, which are characterized by the production of virus-derived small RNAs of 25-29 and 21 nucleotides, respectively. The exo-siRNA pathway is considered to be the key mosquito antiviral response mechanism. In Aedes aegypti-derived cells, Zika virus (ZIKV)-specific siRNAs were produced and loaded into the exo-siRNA pathway effector protein Argonaute 2 (Ago2); although the knockdown of Ago2 did not enhance virus replication. Enhanced ZIKV replication was observed in a Dcr2-knockout cell line suggesting that the exo-siRNA pathway is implicated in the antiviral response. Although ZIKV-specific piRNA-sized small RNAs were detected, these lacked the characteristic piRNA ping-pong signature motif and were bound to Ago3 but not Piwi5 or Piwi6. Silencing of PIWI proteins indicated that the knockdown of Ago3, Piwi5 or Piwi6 did not enhance ZIKV replication and only Piwi4 displayed antiviral activity. We also report that the expression of ZIKV capsid (C) protein amplified the replication of a reporter alphavirus; although, unlike yellow fever virus C protein, it does not inhibit the exo-siRNA pathway. Our findings elucidate ZIKV-mosquito RNAi interactions that are important for understanding its spread.
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http://dx.doi.org/10.1371/journal.pntd.0006010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667879PMC
October 2017

Analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection.

J Gen Virol 2017 Aug 8;98(8):2043-2060. Epub 2017 Aug 8.

MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, Scotland, UK.

Tick-borne encephalitis virus (TBEV) is a member of the genus Flavivirus. It can cause serious infections in humans that may result in encephalitis/meningoencephalitis. Although several studies have described the involvement of specific genes in the host response to TBEV infection in the central nervous system (CNS), the overall network remains poorly characterized. Therefore, we investigated the response of DAOY cells (human medulloblastoma cells derived from cerebellar neurons) to TBEV (Neudoerfl strain, Western subtype) infection to characterize differentially expressed genes by transcriptome analysis. Our results revealed a wide panel of interferon-stimulated genes (ISGs) and pro-inflammatory cytokines, including type III but not type I (or II) interferons (IFNs), which are activated upon TBEV infection, as well as a number of non-coding RNAs, including long non-coding RNAs. To obtain a broader view of the pathways responsible for eliciting an antiviral state in DAOY cells we examined the effect of type I and III IFNs and found that only type I IFN pre-treatment inhibited TBEV production. The cellular response to TBEV showed only partial overlap with gene expression changes induced by IFN-β treatment - suggesting a virus-specific signature - and we identified a group of ISGs that were highly up-regulated following IFN-β treatment. Moreover, a high rate of down-regulation was observed for a wide panel of pro-inflammatory cytokines upon IFN-β treatment. These data can serve as the basis for further studies of host-TBEV interactions and the identification of ISGs and/or lncRNAs with potent antiviral effects in cases of TBEV infection in human neuronal cells.
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http://dx.doi.org/10.1099/jgv.0.000853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817271PMC
August 2017

Understanding the role of microRNAs in the interaction of Aedes aegypti mosquitoes with an insect-specific flavivirus.

J Gen Virol 2017 Jul 12;98(7):1892-1903. Epub 2017 Jul 12.

Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

The Flavivirus genus contains some of the most prevalent vector-borne viruses, such as the dengue, Zika and yellow fever viruses that cause devastating diseases in humans. However, the insect-specific clade of flaviviruses is restricted to mosquito hosts, albeit they have retained the general features of the genus, such as genome structure and replication. The interactions between insect-specific flaviviruses (ISFs) and their mosquito hosts are largely unknown. Pathogenic flaviviruses are known to modulate host-derived microRNAs (miRNAs), a class of non-coding RNAs that are important in controlling gene expression. Alterations in miRNAs may represent changes in host gene expression and promote understanding of virus-host interactions. The role of miRNAs in ISF-mosquito interactions is largely unknown. A recently discovered Australian ISF, Palm Creek virus (PCV), has the ability to suppress medically relevant flaviviruses. Here, we investigated the potential involvement of miRNAs in PCV infection using the model mosquito Aedes aegypti. By combining small-RNA sequencing and bioinformatics analysis, differentially expressed miRNAs were determined. Our results indicated that PCV infection hardly affects host miRNAs. Out of 101 reported miRNAs of Ae. aegypti, only aae-miR-2940-5p had a significantly altered expression over the course of infection. However, further analysis of aae-miR-2940-5p revealed that this miRNA does not have any direct impact on PCV replication in vitro. Thus, overall the results suggest that PCV infection has a limited effect on the mosquito miRNA profile and therefore miRNAs may not play a significant role in the PCV-Ae. aegypti interaction.
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http://dx.doi.org/10.1099/jgv.0.000832DOI Listing
July 2017

Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses.

mSphere 2017 May-Jun;2(3). Epub 2017 May 3.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland.

The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an -derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions.
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http://dx.doi.org/10.1128/mSphere.00144-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415634PMC
May 2017

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

mSphere 2017 May-Jun;2(3). Epub 2017 May 3.

Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany.

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 cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult sp. and mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in sp. but also in mosquitoes, indicating that antiviral RNA interference in mosquitoes is similar to the described activities of RNAi in 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. Rift Valley fever virus (RVFV; , ) 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 . 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 mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.
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http://dx.doi.org/10.1128/mSphere.00090-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415632PMC
May 2017

Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen.

Lancet Infect Dis 2017 04 1;17(4):e107-e117. Epub 2017 Feb 1.

Institute for Glycomics, Griffith University, Gold Coast, QLD, Australia.

Re-emergence of chikungunya virus, a mosquito-transmitted pathogen, is of serious public health concern. In the past 15 years, after decades of infrequent, sporadic outbreaks, the virus has caused major epidemic outbreaks in Africa, Asia, the Indian Ocean, and more recently the Caribbean and the Americas. Chikungunya virus is mainly transmitted by Aedes aegypti mosquitoes in tropical and subtropical regions, but the potential exists for further spread because of genetic adaptation of the virus to Aedes albopictus, a species that thrives in temperate regions. Chikungunya virus represents a substantial health burden to affected populations, with symptoms that include severe joint and muscle pain, rashes, and fever, as well as prolonged periods of disability in some patients. The inflammatory response coincides with raised levels of immune mediators and infiltration of immune cells into infected joints and surrounding tissues. Animal models have provided insights into disease pathology and immune responses. Although host innate and adaptive responses have a role in viral clearance and protection, they can also contribute to virus-induced immune pathology. Understanding the mechanisms of host immune responses is essential for the development of treatments and vaccines. Inhibitory compounds targeting key inflammatory pathways, as well as attenuated virus vaccines, have shown some success in animal models, including an attenuated vaccine strain based on an isolate from La Reunion incorporating an internal ribosome entry sequence that prevents the virus from infecting mosquitoes and a vaccine based on virus-like particles expressing envelope proteins. However, immune correlates of protection, as well as the safety of prophylactic and therapeutic candidates, are important to consider for their application in chikungunya infections. In this Review, we provide an update on chikungunya virus with regard to its epidemiology, molecular virology, virus-host interactions, immunological responses, animal models, and potential antiviral therapies and vaccines.
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http://dx.doi.org/10.1016/S1473-3099(16)30385-1DOI Listing
April 2017

The Antiviral RNAi Response in Vector and Non-vector Cells against Orthobunyaviruses.

PLoS Negl Trop Dis 2017 01 6;11(1):e0005272. Epub 2017 Jan 6.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: Vector arthropods control arbovirus replication and spread through antiviral innate immune responses including RNA interference (RNAi) pathways. Arbovirus infections have been shown to induce the exogenous small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) pathways, but direct antiviral activity by these host responses in mosquito cells has only been demonstrated against a limited number of positive-strand RNA arboviruses. For bunyaviruses in general, the relative contribution of small RNA pathways in antiviral defences is unknown.

Methodology/principal Findings: The genus Orthobunyavirus in the Bunyaviridae family harbours a diverse range of mosquito-, midge- and tick-borne arboviruses. We hypothesized that differences in the antiviral RNAi response in vector versus non-vector cells may exist and that could influence viral host range. Using Aedes aegypti-derived mosquito cells, mosquito-borne orthobunyaviruses and midge-borne orthobunyaviruses we showed that bunyavirus infection commonly induced the production of small RNAs and the effects of the small RNA pathways on individual viruses differ in specific vector-arbovirus interactions.

Conclusions/significance: These findings have important implications for our understanding of antiviral RNAi pathways and orthobunyavirus-vector interactions and tropism.
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http://dx.doi.org/10.1371/journal.pntd.0005272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245901PMC
January 2017

Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

PLoS Negl Trop Dis 2016 Oct 5;10(10):e0005048. Epub 2016 Oct 5.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions.

Methodology/principal Findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action.

Conclusions/significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.
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http://dx.doi.org/10.1371/journal.pntd.0005048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5051680PMC
October 2016

Wolbachia restricts insect-specific flavivirus infection in Aedes aegypti cells.

J Gen Virol 2016 Nov 28;97(11):3024-3029. Epub 2016 Sep 28.

MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, Scotland, UK.

Mosquito-borne viruses are known to cause disease in humans and livestock and are often difficult to control due to the lack of specific antivirals and vaccines. The Wolbachia endosymbiont has been widely studied for its ability to restrict positive-strand RNA virus infection in mosquitoes, although little is known about the precise antiviral mechanism. In recent years, a variety of insect-specific viruses have been discovered in mosquitoes and an interaction with mosquito-borne viruses has been reported for some of them; however, nothing is known about the effect of Wolbachia on insect-specific virus infection in mosquitoes. Here, we show that transinfection of the Drosophila-derived wMelPop Wolbachia strain into Aedes aegypti-derived cells resulted in inhibition and even clearance of the persistent cell-fusing agent flavivirus infection in these cells. This broadens the antiviral activity of Wolbachia from acute infections to persistent infections and from arboviruses to mosquito-specific viruses. In contrast, no effect on the Phasi Charoen-like bunyavirus persistent infection in these cells was observed, suggesting a difference in Wolbachia inhibition between positive- and negative-strand RNA viruses.
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http://dx.doi.org/10.1099/jgv.0.000617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120408PMC
November 2016

Advancing vector biology research: a community survey for future directions, research applications and infrastructure requirements.

Pathog Glob Health 2016 Jun-Jul;110(4-5):164-72. Epub 2016 Aug 11.

k Department of Parasites and Insect Vectors , Institut Pasteur, Unit of Insect Vector Genetics and Genomics , Paris cedex 15 , France.

Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target transmission by the vector. While insecticides are an important part of this arsenal, appearance of resistance mechanisms is increasingly common. Novel tools for genetic manipulation of vectors, use of Wolbachia endosymbiotic bacteria, and other biological control mechanisms to prevent pathogen transmission have led to promising new intervention strategies, adding to strong interest in vector biology and genetics as well as vector-pathogen interactions. Vector research is therefore at a crucial juncture, and strategic decisions on future research directions and research infrastructure investment should be informed by the research community. A survey initiated by the European Horizon 2020 INFRAVEC-2 consortium set out to canvass priorities in the vector biology research community and to determine key activities that are needed for researchers to efficiently study vectors, vector-pathogen interactions, as well as access the structures and services that allow such activities to be carried out. We summarize the most important findings of the survey which in particular reflect the priorities of researchers in European countries, and which will be of use to stakeholders that include researchers, government, and research organizations.
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http://dx.doi.org/10.1080/20477724.2016.1211475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072118PMC
April 2017

The Importance of Socio-Economic Versus Environmental Risk Factors for Reported Dengue Cases in Java, Indonesia.

PLoS Negl Trop Dis 2016 09 7;10(9):e0004964. Epub 2016 Sep 7.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: Dengue is a major mosquito-borne viral disease and an important public health problem. Identifying which factors are important determinants in the risk of dengue infection is critical in supporting and guiding preventive measures. In South-East Asia, half of all reported fatal infections are recorded in Indonesia, yet little is known about the epidemiology of dengue in this country.

Methodology/principal Findings: Hospital-reported dengue cases in Banyumas regency, Central Java were examined to build Bayesian spatial and spatio-temporal models assessing the influence of climatic, demographic and socio-economic factors on the risk of dengue infection. A socio-economic factor linking employment type and economic status was the most influential on the risk of dengue infection in the Regency. Other factors such as access to healthcare facilities and night-time temperature were also found to be associated with higher risk of reported dengue infection but had limited explanatory power.

Conclusions/significance: Our data suggest that dengue infections are triggered by indoor transmission events linked to socio-economic factors (employment type, economic status). Preventive measures in this area should therefore target also specific environments such as schools and work areas to attempt and reduce dengue burden in this community. Although our analysis did not account for factors such as variations in immunity which need further investigation, this study can advise preventive measures in areas with similar patterns of reported dengue cases and environment.
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http://dx.doi.org/10.1371/journal.pntd.0004964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014450PMC
September 2016

Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection.

Immunity 2016 06;44(6):1455-69

Virus Host Interaction Team, Section of Infection and Immunity, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds LS9 7TF, UK. Electronic address:

Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.
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http://dx.doi.org/10.1016/j.immuni.2016.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920956PMC
June 2016

Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

PLoS Pathog 2016 Apr 18;12(4):e1005536. Epub 2016 Apr 18.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced) mechanism.
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http://dx.doi.org/10.1371/journal.ppat.1005536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835223PMC
April 2016

Dengue in Java, Indonesia: Relevance of Mosquito Indices as Risk Predictors.

PLoS Negl Trop Dis 2016 Mar 11;10(3):e0004500. Epub 2016 Mar 11.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: No vaccine is currently available for dengue virus (DENV), therefore control programmes usually focus on managing mosquito vector populations. Entomological surveys provide the most common means of characterising vector populations and predicting the risk of local dengue virus transmission. Despite Indonesia being a country strongly affected by DENV, only limited information is available on the local factors affecting DENV transmission and the suitability of available survey methods for assessing risk.

Methodology/principal Findings: We conducted entomological surveys in the Banyumas Regency (Central Java) where dengue cases occur on an annual basis. Four villages were sampled during the dry and rainy seasons: two villages where dengue was endemic, one where dengue cases occurred sporadically and one which was dengue-free. In addition to data for conventional larvae indices, we collected data on pupae indices, and collected adult mosquitoes for species identification in order to determine mosquito species composition and population density. Traditionally used larval indices (House indices, Container indices and Breteau indices) were found to be inadequate as indicators for DENV transmission risk. In contrast, species composition of adult mosquitoes revealed that competent vector species were dominant in dengue endemic and sporadic villages.

Conclusions/significance: Our data suggested that the utility of traditional larvae indices, which continue to be used in many dengue endemic countries, should be re-evaluated locally. The results highlight the need for validation of risk indicators and control strategies across DENV affected areas here and perhaps elsewhere in SE Asia.
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http://dx.doi.org/10.1371/journal.pntd.0004500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788303PMC
March 2016

Modulation of Potassium Channels Inhibits Bunyavirus Infection.

J Biol Chem 2016 Feb 16;291(7):3411-22. Epub 2015 Dec 16.

From the School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT,

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.
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http://dx.doi.org/10.1074/jbc.M115.692673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751384PMC
February 2016

A novel insect-specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes vigilax populations in Sydney, Australia.

Virology 2015 Dec 27;486:272-83. Epub 2015 Oct 27.

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Queensland, Australia. Electronic address:

To date, insect-specific flaviviruses (ISFs) have only been isolated from mosquitoes and increasing evidence suggests that ISFs may affect the transmission of pathogenic flaviviruses. To investigate the diversity and prevalence of ISFs in Australian mosquitoes, samples from various regions were screened for flaviviruses by ELISA and RT-PCR. Thirty-eight pools of Aedes vigilax from Sydney in 2007 yielded isolates of a novel flavivirus, named Parramatta River virus (PaRV). Sequencing of the viral RNA genome revealed it was closely related to Hanko virus with 62.3% nucleotide identity over the open reading frame. PaRV failed to grow in vertebrate cells, with only Aedes-derived mosquito cell lines permissive to replication, suggesting a narrow host range. 2014 collections revealed that PaRV had persisted in A. vigilax populations in Sydney, with 88% of pools positive. Further investigations into its mode of transmission and potential to influence vector competence of A. vigilax for pathogenic viruses are warranted.
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http://dx.doi.org/10.1016/j.virol.2015.07.021DOI Listing
December 2015
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