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Culicoides midge bites modulate the host response and impact on bluetongue virus infection in sheep.

ABSTRACT: Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.

SUBMITTER: Pages N

PROVIDER: S-EPMC3885445 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Culicoides midge bites modulate the host response and impact on bluetongue virus infection in sheep.

Pages Nonito N Bréard Emmanuel E Urien Céline C Talavera Sandra S Viarouge Cyril C Lorca-Oro Cristina C Jouneau Luc L Charley Bernard B Zientara Stéphan S Bensaid Albert A Solanes David D Pujols Joan J Schwartz-Cornil Isabelle I

PloS one 20140108 1

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia a ...[more]

PMID: 24421899

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Project description:BackgroundThe new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV.ResultsOur analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study.ConclusionsThe reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Project description:UnlabelledBluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence.ImportanceBluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

Project description:Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ?10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.

Project description:BackgroundHaemoproteus parasites are widespread, and some species cause disease in wild and domestic birds. However, the insect vectors remain unknown for the majority of species and genetic lineages of avian Haemoproteus. This information is crucial for better understanding the biology of haemoproteids, the epidemiology of haemoproteosis, and the development of morphological characters of sporogonic stages in wildlife haemosporidian parasites. It remains unclear whether the specificity of Haemoproteus parasites for vectors is broad or the transmission of a given parasite can be restricted to a single or few species of vectors. The aim of this study was to examine the sporogonic development of four species of common European avian haemoproteids in the common biting midge Culicoides impunctatus.MethodsWild-caught females of C. impunctatus were infected experimentally by allowing them to take blood meals on naturally infected Muscicapa striata, Cyanistes caeruleus, Ficedula hypoleuca and Motacilla flava harbouring mature gametocytes of Haemoproteus balmorali (genetic lineage hSFC9), H. majoris (hPARUS1), H. motacillae (hYWT1) and H. pallidus (hPFC1), respectively. Infected insects were collected, maintained under laboratory conditions and dissected daily in order to detect the development of ookinetes, oocysts and sporozoites. Microscopic examination and polymerase chain reaction based methods were used to detect the parasites. Bayesian analysis was applied to identify phylogenetic relationships among Haemoproteus lineages.ResultsAll investigated parasites completed sporogony in C. impunctatus, indicating broad susceptibility of this biting midge for numerous Haemoproteus parasites. Ookinetes, oocysts and sporozoites were reported, described and compared morphologically. The investigated parasite species can be distinguished at the sporogony stage, particularly with regards to the morphology and rate of development of mature ookinetes. Analysis of data from the literature, and this study, shows that 12 genetically distantly related Haemoproteus parasites complete sporogony in C. impunctatus.ConclusionsSusceptibility of C. impunctatus is broad for Haemoproteus parasites, indicating that this biting midge is an important natural vector of numerous species of avian haemoproteids in Europe. Some Haemoproteus species can be readily distinguished using morphological characters of ookinetes and sporozoites, as well as the rate of ookinete development. These characters can be used for the identification of Haemoproteus species during sporogony in vectors, and are worth more attention in these parasite taxonomy studies at the species levels.

Project description:Bluetongue virus (BTV), the prototypical Orbivirus that belongs to the Sedoreoviridae family, is transmitted by the bite of infected Culicoides midges and affects domestic and wild ruminants producing great economic losses. The infection induces an IFN response, followed by an adaptive immune response that plays a critical role in disease clearance. BTV can nonetheless impair IFN and humoral responses. The main goal of this study was to gain a more detailed understanding of BTV pathogenesis and its effects on immune cell populations. To this end, we combined flow cytometry and transcriptomic analyses of several immune cells at different times post-infection (pi). Four sheep were infected with BTV serotype 8 and blood samples collected at days 0, 3, 7 and 15pi to isolate peripheral blood mononuclear cells. Transcriptomic analysis of B-cell marker+, CD4+, CD8+, and CD14+ sorted cells showed that the maximum number of differentially expressed genes occurred at day 7pi, which coincided with the peak of infection. KEGG pathway enrichment analysis in B-cell marker+, CD4+, and CD14+ cells indicated that genes belonging to virus sensing and immune response initiation pathways were enriched and day 3 and 7 pi. Transcriptomic analysis also showed that T cell exhaustion pathway was enriched in CD4+ cells at day 7pi, while CD8+ cells showed downregulated immune response initiation pathways at this timepoint. When T cell functionality was assessed, ELISpot assays, intracellular cytokine staining, and proliferation assays demonstrated that BTV produced an acute inhibition of CD4+ and CD8+ T cell activation at the peak of replication. Furthermore, this coincided with PD-L1 upregulation on the surface of CD4+ and CD8+ T cells as well as monocytes. Taken together, these data indicate that BTV exploits the PD1/PD-L1 immune checkpoint to impair T cell responses. These findings identify several mechanisms in the interaction between host and BTV, which could help develop better tools to combat the disease.

Dataset Information

Culicoides midge bites modulate the host response and impact on bluetongue virus infection in sheep.

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Culicoides midge bites modulate the host response and impact on bluetongue virus infection in sheep.

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