Jean-Bernard Duchemin

Secreted Vago restricts West Nile virus infection in Culex mosquito cells by activating the Jak-STAT pathway

Although West Nile virus (WNV) and other arthropod-borne viruses are a major public health problem, the mechanisms of antiviral immunity in mosquitoes are poorly understood. Dicer-2, responsible for the RNAi-mediated response through the C-terminal RNase-III domain, also contains an N-terminal DExD/H-box helicase domain similar to mammalian RIG-I/MDA5 which, in Drosophila, was found to be required for activation of an antiviral gene, Vago. Here we show that the Culex orthologue of Vago (CxVago) is up-regulated in response to WNV infection in a Dicer-2–dependent manner. Further, our data show that CxVago is a secreted peptide that restricts WNV infection by activation of the Jak-STAT pathway. Thus, Vago appears to function as an IFN-like antiviral cytokine in mosquitoes.

Dicer-2-Dependent Activation of Culex Vago Occurs via the TRAF-Rel2 Signaling Pathway

Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that Culex Vago (CxVago), which is induced and secreted from West Nile virus (WNV)-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation. Using a luciferase reporter assay, we show that a NF-κB-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-κB ortholog, Rel2, plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of CxVago.

Detection of Low-Level Cardinium and Wolbachia Infections in Culicoides

ABSTRACT Bacterial endosymbionts have been identified as potentially useful biological control agents for a range of invertebrate vectors of disease. Previous studies of Culicoides (Diptera: Ceratopogonidae) species using conventional PCR assays have provided evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20 species of Culicoides for Wolbachia and Cardinium, utilizing a combination of conventional PCR and more sensitive quantitative PCR (qPCR) assays. Low levels of Cardinium DNA were detected in females of all but one of the Culicoides species screened, and low levels of Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence analysis based on partial 16S rRNA gene and gyrB sequences identified “Candidatus Cardinium hertigii” from group C, which has previously been identified in Culicoides from Japan, Israel, and the United Kingdom. Wolbachia strains detected in this study showed 98 to 99% sequence identity to Wolbachia previously detected from Culicoides based on the 16S rRNA gene, whereas a strain with a novel wsp sequence was identified in Culicoides narrabeenensis. Cardinium isolates grouped to geographical regions independent of the host Culicoides species, suggesting possible geographical barriers to Cardinium movement. Screening also identified Asaia bacteria in Culicoides. These findings point to a diversity of low-level endosymbiont infections in Culicoides, providing candidates for further characterization and highlighting the widespread occurrence of these endosymbionts in this insect group.

Seasonal drivers of the epidemiology of arthropod-borne viruses in Australia

Arthropod-borne viruses are a major cause of emerging disease with significant public health and economic impacts. However, the factors that determine their activity and seasonality are not well understood. In Australia, a network of sentinel cattle herds is used to monitor the distribution of several such viruses and to define virus-free regions. Herein, we utilize these serological data to describe the seasonality, and its drivers, of three economically important animal arboviruses: bluetongue virus, Akabane virus and bovine ephemeral fever virus. Through epidemiological time-series analyses of sero-surveillance data of 180 sentinel herds between 2004–2012, we compared seasonal parameters across latitudes, ranging from the tropical north (−10°S) to the more temperate south (−40°S). This analysis revealed marked differences in seasonality between distinct geographic regions and climates: seasonality was most pronounced in southern regions and gradually decreased as latitude decreased toward the Equator. Further, we show that both the timing of epidemics and the average number of seroconversions have a strong geographical component, which likely reflect patterns of vector abundance through co-varying climatic factors, especially temperature and rainfall. Notably, despite their differences in biology, including insect vector species, all three viruses exhibited very similar seasonality. By revealing the factors that shape spatial and temporal distributions, our study provides a more complete understanding of arbovirus seasonality that will enable better risk predictions.

Zika vector transmission risk in temperate Australia: a vector competence study

BackgroundZika virus is an emerging pathogen of global importance. It has been responsible for recent outbreaks in the Americas and in the Pacific region. This study assessed five different mosquito species from the temperate climatic zone in Australia and included Aedes albopictus as a potentially invasive species.MethodsMosquitoes were orally challenged by membrane feeding with Zika virus strain of Cambodia 2010 origin, belonging to the Asian clade. Virus infection and dissemination were assessed by quantitative PCR on midgut and carcass after dissection. Transmission was assessed by determination of cytopathogenic effect of saliva (CPE) on Vero cells, followed by determination of 50% tissue culture infectious dose (TCID50) for CPE positive samples. Additionally, the presence of Wolbachia endosymbiont infection was assessed by qPCR and standard PCR.ResultsCulex mosquitoes were found unable to present Zika virus in saliva, as demonstrated by molecular as well as virological methods. Aedes aegypti, was used as a positive control for Zika infection and showed a high level of virus infection, dissemination and transmission. Local Aedes species, Ae. notoscriptus and, to a lesser degree, Ae. camptorhynchus were found to expel virus in their saliva and contained viral nucleic acid within the midgut. Molecular assessment identified low or no dissemination for these species, possibly due to low virus loads. Ae. albopictus from Torres Strait islands origin was shown as an efficient vector. Cx quinquefasciatus was shown to harbour Wolbachia endosymbionts at high prevalence, whilst no Wolbachia was found in Cx annulirostris. The Australian Ae. albopictus population was shown to harbour Wolbachia at high frequency.ConclusionsThe risk of local Aedes species triggering large Zika epidemics in the southern parts of Australia is low. The potentially invasive Ae. albopictus showed high prevalence of virus in the saliva and constitutes a potential threat if this mosquito species becomes established in mainland Australia. Complete risk analysis of Zika transmission in the temperate zone would require an assessment of the impact of temperature on Zika virus replication within local and invasive mosquito species.

Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes

Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

Longitudinal follow‐up of malaria transmission dynamics in two villages in a Sahelian area of Niger during a nationwide insecticide‐treated bednet distribution programme

Malaria transmission was monitored in two villages in the Sahel zone of Niger over 4 years. During this period, a nationwide vector control programme was carried out in which insecticide‐treated bednets were distributed free to mothers of children aged <5 years. Anopheles gambiae and Anopheles arabiensis (Diptera: Culicidae) were found to be the major malaria vectors. The dynamics of An. gambiae s.l. did not vary dramatically over the study period although the proportion of female mosquitoes found resting indoors decreased in both villages and, in one village, the parity rate and sporozoite index were significantly reduced after bednet distribution. By contrast with An. gambiae, the dynamics of Anopheles funestus altered greatly after the bednet distribution period, when adult density, endophagous rate and sporozoite rates decreased dramatically. Our observations highlight the importance of quantifying and monitoring the dynamics and infections of malaria vectors during large‐scale vector control interventions.

Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera: Ceratopogonidae), using multi-locus DNA microsatellites.

Bluetongue virus (BTV) is a major pathogen of ruminants that is transmitted by biting midges (Culicoides spp.). Australian BTV serotypes have origins in Asia and are distributed across the continent into two distinct episystems, one in the north and another in the east. Culicoides brevitarsis is the major vector of BTV in Australia and is distributed across the entire geographic range of the virus. Here, we describe the isolation and use of DNA microsatellites and gauge their ability to determine population genetic connectivity of C. brevitarsis within Australia and with countries to the north. Eleven DNA microsatellite markers were isolated using a novel genomic enrichment method and identified as useful for genetic analyses of sampled populations in Australia, northern Papua New Guinea (PNG) and Timor-Leste. Significant (Pu2009<u20090.05) population genetic subdivision was observed between all paired regions, though the highest levels of genetic sub-division involved pair-wise tests with PNG (PNG vs. Australia (FSTu2009=u20090.120) and PNG vs. Timor-Leste (FSTu2009=u20090.095)). Analysis of multi-locus allelic distributions using STRUCTURE identified a most probable two-cluster population model, which separated PNG specimens from a cluster containing specimens from Timor-Leste and Australia. The source of incursions of this species in Australia is more likely to be Timor-Leste than PNG. Future incursions of BTV positive C. brevitarsis into Australia may be genetically identified to their source populations using these microsatellite loci. The vector’s panmictic genetic structure within Australia cannot explain the differential geographic distribution of BTV serotypes.

Neurophysiology of Insects Using Microelectrode Arrays: Current Trends and Future Prospects

Simple to complex behaviors are directed by the brain, which possess nervous cells, called neurons. Mammals have billions of neurons, organized in networks, making their study difficult. Although methods have well evolved since the last century, studying a simpler model is the key to resolving neuronal communication. In this review, we demonstrate that insects are an excellent model and tool to understand neural mechanisms. Moreover, new technology, such as Microelectrodes Arrays (MEAs), is an innovative method which opens the possibility to study neuron clusters, rather than individual cells. A combined method of an insect model and MEAs technology may lead to great discoveries in neurophysiology, advancing progress in pharmacology, infectious and neurodegenerative diseases, agriculture maintenance and robotics.

Neurotropism and behavioral changes associated with Zika infection in the vector Aedes aegypti

Understanding Zika virus infection dynamics is essential, as its recent emergence revealed possible devastating neuropathologies in humans, thus causing a major threat to public health worldwide. Recent research allowed breakthrough in our understanding of the virus and host pathogenesis; however, little is known on its impact on its main vector, Aedes aegypti. Here we show how Zika virus targets Aedes aegypti’s neurons and induces changes in its behavior. Results are compared to dengue virus, another flavivirus, which triggers a different pattern of behavioral changes. We used microelectrode array technology to record electrical spiking activity of mosquito primary neurons post infections and discovered that only Zika virus causes an increase in spiking activity of the neuronal network. Confocal microscopy also revealed an increase in synapse connections for Zika virus-infected neuronal networks. Interestingly, the results also showed that mosquito responds to infection by overexpressing glutamate regulatory genes while maintaining virus levels. This neuro-excitation, possibly via glutamate, could contribute to the observed behavioral changes in Zika virus-infected Aedes aegypti females. This study reveals the importance of virus-vector interaction in arbovirus neurotropism, in humans and vector. However, it appears that the consequences differ in the two hosts, with neuropathology in human host, while behavioral changes in the mosquito vector that may be advantageous to the virus.