- Volume 1, Issue 10, 2019
Volume 1, Issue 10, 2019
- Abstracts from the International Meeting on Arboviruses and their Vectors 2019
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- Poster Abstract
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Biotyping of TBEV-infected IRE/CTVM19 tick cell line
Background. Ticks have developed defense mechanisms and pathways against transmitted infections, including tick-borne encephalitis virus (TBEV). An important issue is to reveal mechanisms allowing them to control the virus at a level which does not hinder ticks’ fitness and development.
Methods. Biotyping was performed on an Autoflex Speed MALDI-TOF/TOF (Bruker Daltonik). Protein digests were analyzed using Synapt G2-Si High Definition mass spectrometer (Waters).
Results. MS profiles of TBEV-infected and non-infected IRE/CTVM19 cells were analyzed using principal component analysis. Obtained spectra were clustered based on the cultivation time, but not the infection status. Nevertheless, analysis of loading plots revealed different factors to be important for clustering of infected and non-infected cells. Out of them, nine were assigned with proteins: five and four for non-infected and infected cells, respectively. Peak with m/z 8565 was found to be of interest from viewpoint of tick-virus interaction and assigned to proteasome subunit alpha type (B7QE67).
Conclusion. MALDI-TOF MS was shown to be useful for characterization of tick cell lines and studying tick-virus interactions. Signals in MS profiles discriminating cell aging and those affected by TBEV were revealed, and matched with proteins.
We thank Dr Lesley Bell-Sakyi and the Tick Cell Biobank for provision of IRE/CTVM19 cells.
This study was supported by the MŠMT ČR INTER-ACTION project (LTARF 18021); GAČR (18-27204S), European Regional Development Fund Project (CZ.02.1.01/0.0/0.0/15_003/0000441), and MSHE RF (#14.616.21.0094, RFMEFI61618X0094). Access to instruments and other facilities was supported by the Czech research infrastructure for systems biology C4SYS (LM2015055).
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Chikungunya virus resistant to the antiviral favipiravir is severely attenuated in mosquitoes
It is currently unclear whether antiviral drug-resistant arboviruses can be transmitted by their mosquito vectors. We showed previously that the dissemination and the transmission of a chikungunya virus (CHIKV) resistant to the antiviral favipiravir was markedly decreased as compared to WT (Delang et al, mSphere 2018). The attenuated phenotype of this resistant virus was confirmed in Aag2 and C6/36 mosquito cells. Here, we aimed to study the mechanism of the attenuated phenotype in more detail.
First, replication kinetics studies at 32°C for both Vero and mosquito cells confirmed that the attenuated fitness in mosquito cells is associated with the cell line and not with temperature. A passaging experiment of WT CHIKV on Vero cells in the absence of favipiravir showed that the observed attenuation of the resistant CHIKV was not due to passaging on Vero cells during the resistance selection. To identify the molecular mechanism of the attenuated phenotype, the genes encoding for the non-structural proteins (nsP) of the favipiravirres CHIKV were swapped into a WT CHIKV backbone. The replication fitness of these nsP2, nsP3 or nsP4 single swap variants did not differ significantly from the fitness of WT CHIKV in Vero and C6/36 cells, indicating that a combination of mutations in multiple non-structural proteins is responsible for the attenuated phenotype. Double nsP swap variants are therefore constructed and these will provide additional information on the molecular mechanism of the attenuation. Together, our results may provide interesting insights in the mosquito tropism of CHIKV.
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Chikungunya virus resistant to the antiviral favipiravir is severely attenuated in mosquitoes
It is currently unclear whether antiviral drug-resistant arboviruses can be transmitted by their mosquito vectors. We showed previously that the dissemination and the transmission of a chikungunya virus (CHIKV) resistant to the antiviral favipiravir was markedly decreased as compared to WT (Delang et al, mSphere 2018). The attenuated phenotype of this resistant virus was confirmed in Aag2 and C6/36 mosquito cells. Here, we aimed to study the mechanism of the attenuated phenotype in more detail.
First, replication kinetics studies at 32°C for both Vero and mosquito cells confirmed that the attenuated fitness in mosquito cells is associated with the cell line and not with temperature. A passaging experiment of WT CHIKV on Vero cells in the absence of favipiravir showed that the observed attenuation of the resistant CHIKV was not due to passaging on Vero cells during the resistance selection. To identify the molecular mechanism of the attenuated phenotype, the genes encoding for the non-structural proteins (nsP) of the favipiravirres CHIKV were swapped into a WT CHIKV backbone. The replication fitness of these nsP2, nsP3 or nsP4 single swap variants did not differ significantly from the fitness of WT CHIKV in Vero and C6/36 cells, indicating that a combination of mutations in multiple non-structural proteins is responsible for the attenuated phenotype. Double nsP swap variants are therefore constructed and these will provide additional information on the molecular mechanism of the attenuation. Together, our results may provide interesting insights in the mosquito tropism of CHIKV.
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Lumpy skin disease virus does not replicate productively in insect cell lines
Lumpy skin disease virus (LSDV) is a capripoxvirus of cattle causing a severe disease that results in substantial economic impact to affected communities. The virus is endemic across sub-Saharan Africa, and has recently entered Europe and the Balkans. The virus is thought to be transmitted by an insect vector, but little is known about the role of the vector in the LSDV cycle.
In order to investigate interactions between LSDV and insect vectors this work studied the permissivity of insect cell lines for LSDV. Insect cell lines were inoculated with LSDV strain Cameroon at a multiplicity of infection (MOI) of 5. The mammalian cell line MDBK was infected as a positive control. Samples were collected up to a week after infection and virus amounts measured using titrated plaque assays in order to construct a growth curve.
Lumpy skin disease virus replicated in the MDBK mammalian cell line, increasing by 3log10 over the 7 day incubation from 2×104 pfu/ml to 2×107 pfu/ml. In contrast, no replication of LSDV was detected in the insect cell lines.
This work shows that LSDV does not productively replicate in insect cell lines and supports the current hypothesis that the insects act as mechanical rather than biological vectors of the virus.
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Lumpy skin disease virus does not replicate productively in insect cell lines
Lumpy skin disease virus (LSDV) is a capripoxvirus of cattle causing a severe disease that results in substantial economic impact to affected communities. The virus is endemic across sub-Saharan Africa, and has recently entered Europe and the Balkans. The virus is thought to be transmitted by an insect vector, but little is known about the role of the vector in the LSDV cycle.
In order to investigate interactions between LSDV and insect vectors this work studied the permissivity of insect cell lines for LSDV. Insect cell lines were inoculated with LSDV strain Cameroon at a multiplicity of infection (MOI) of 5. The mammalian cell line MDBK was infected as a positive control. Samples were collected up to a week after infection and virus amounts measured using titrated plaque assays in order to construct a growth curve.
Lumpy skin disease virus replicated in the MDBK mammalian cell line, increasing by 3log10 over the 7 day incubation from 2×104 pfu/ml to 2×107 pfu/ml. In contrast, no replication of LSDV was detected in the insect cell lines.
This work shows that LSDV does not productively replicate in insect cell lines and supports the current hypothesis that the insects act as mechanical rather than biological vectors of the virus.
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Posaconazole is a novel inhibitor for alphavirus viral entry
Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes millions of infection globally. Posaconazole (PCZ) is an antifungal drug, which we and others have previously found to inhibit replication of a number of viruses, including dengue virus, a member of the Flaviviridae family. In this study, we analyzed the antiviral activity of PCZ against alphaviruses. We found that PCZ potently inhibits a number of alphaviruses, including Semliki forest virus (SFV), Sindbis virus (SINV) and CHIKV with half maximal effective concentration (EC50) of 2.3 μM, 4.0 μM and 0.8 μM, respectively. Time-of-addition assays indicated that PCZ treatment before and at the time of SFV infection showed potent inhibition, whereas addition of PCZ at later time points post infection showed minor to no inhibition, suggesting inhibition at an early stage of the replication cycle. In accordance, PCZ treatment of a temperature sensitive mutant of SFV that is capable of cell entry and translation, but not RNA replication, resulted in an almost 90% reduction in luciferase activity. To confirm these findings, PCZ resistant mutant virus were generated and we identified mutations in E1 (V148A) and E2 (H255R) viral glycoproteins, of which the E2 mutation confers partial resistance to PCZ when introduced into wild-type SFV. To see whether PCZ alters clathrin-mediated endocytosis, we analyzed the uptake of fluorescence-tagged transferrin and found that PCZ reduced transferrin uptake by 50% compared to DMSO-treated cells. Together, these results establish PCZ as a novel inhibitor of alphaviruses and identify viral entry as its target.
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Posaconazole is a novel inhibitor for alphavirus viral entry
Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes millions of infection globally. Posaconazole (PCZ) is an antifungal drug, which we and others have previously found to inhibit replication of a number of viruses, including dengue virus, a member of the Flaviviridae family. In this study, we analyzed the antiviral activity of PCZ against alphaviruses. We found that PCZ potently inhibits a number of alphaviruses, including Semliki forest virus (SFV), Sindbis virus (SINV) and CHIKV with half maximal effective concentration (EC50) of 2.3 μM, 4.0 μM and 0.8 μM, respectively. Time-of-addition assays indicated that PCZ treatment before and at the time of SFV infection showed potent inhibition, whereas addition of PCZ at later time points post infection showed minor to no inhibition, suggesting inhibition at an early stage of the replication cycle. In accordance, PCZ treatment of a temperature sensitive mutant of SFV that is capable of cell entry and translation, but not RNA replication, resulted in an almost 90% reduction in luciferase activity. To confirm these findings, PCZ resistant mutant virus were generated and we identified mutations in E1 (V148A) and E2 (H255R) viral glycoproteins, of which the E2 mutation confers partial resistance to PCZ when introduced into wild-type SFV. To see whether PCZ alters clathrin-mediated endocytosis, we analyzed the uptake of fluorescence-tagged transferrin and found that PCZ reduced transferrin uptake by 50% compared to DMSO-treated cells. Together, these results establish PCZ as a novel inhibitor of alphaviruses and identify viral entry as its target.
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Karyotype changes in cultivated tick cell lines
Background. Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and different aspects of tick physiology. However, long-term cultivation of tick cells can influence genome stability. The aim of our work was to analyze and compare the karyotypes of Ixodes scapularis, I. ricinus, and Ornithodoros moubata tick cell lines after long-term been in culture.
Methods. Mitotic spreads were prepared to count the number of chromosomes in ISE6, ISE18, IRE11, IRE/CTVM19, IRE/CTVM20, and OME/CTVM22 cell lines. The genome size of tick cell cultures was estimated by flow cytometry using propidium iodide staining.
Results. The modal chromosome numbers around 22 and around 48 were typical for both I. ricinus and I. scapularis cell lines and differed from the diploid chromosome number in Ixodesticks – 28. In the OME/CTVM22cell line, the modal chromosome number was 33, instead of 20 in Ornithodoros ticks. All tick cell lines had a larger genome size in compare to genomes of parental ticks.
Conclusions.Tick cell lines can be used for research purposes, however, differences in the internal processes between different cell populations should be taken into account.
Acknowledgments: We thank Lesley Bell-Sakyi and the Tick Cell Biobank for provision of the tick cell lines.
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Karyotype changes in cultivated tick cell lines
Background. Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and different aspects of tick physiology. However, long-term cultivation of tick cells can influence genome stability. The aim of our work was to analyze and compare the karyotypes of Ixodes scapularis, I. ricinus, and Ornithodoros moubata tick cell lines after long-term been in culture.
Methods. Mitotic spreads were prepared to count the number of chromosomes in ISE6, ISE18, IRE11, IRE/CTVM19, IRE/CTVM20, and OME/CTVM22 cell lines. The genome size of tick cell cultures was estimated by flow cytometry using propidium iodide staining.
Results. The modal chromosome numbers around 22 and around 48 were typical for both I. ricinus and I. scapularis cell lines and differed from the diploid chromosome number in Ixodesticks – 28. In the OME/CTVM22cell line, the modal chromosome number was 33, instead of 20 in Ornithodoros ticks. All tick cell lines had a larger genome size in compare to genomes of parental ticks.
Conclusions.Tick cell lines can be used for research purposes, however, differences in the internal processes between different cell populations should be taken into account.
Acknowledgments: We thank Lesley Bell-Sakyi and the Tick Cell Biobank for provision of the tick cell lines.
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Targeting functional RNA structures in CHIKV and ZIKV
Emerging arboviruses such as Zika virus (ZIKV) and Chikungunya virus (CHIKV) represent a significant threat to human health and have a high potential to cause outbreaks in the near future. At present, there are no specific antivirals available for either of these important pathogens, despite the wide global prevalence of their vector, Aedes spp. mosquitos.
The positive-strand genomes of ZIKV and CHIKV, members of the flavivirus and alphavirus genera respectively, contain functional, structured cis-acting RNA elements which are essential for virus replication. By specifically targeting such RNA elements using antisense locked nucleic acid oligonucleotides (antisense-LNA), we aim to disrupt their function, and analyse the effect this has on virus replication at different life cycle stages.
Obtaining high quality, single-nucleotide-resolution structural data is essential prior to targeting RNA structures. Consequently, we mapped RNA structural elements within the ZIKV 5’ genome region using a combination of biochemical SHAPE probing, thermodynamic models and phylogenetic analysis. We are currently validating our structural data by analysis of mutant phenotypes in a reverse genetic system.
We demonstrate that functional RNA elements in CHIKV can be specifically targeted - inhibiting replication in both sub-genomic replicon and infectious virus systems. Surface plasmon resonance confirmed that an antisense-LNA binds to a specific stem-loop target with a Kd of 310nM and has an IC50 of 35nM in a sub-genomic replicon system. In future work, we aim to investigate selection of RNA-aptamers against CHIKV and target ZIKV genomic stem-loops using antisense-LNAs.
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Targeting functional RNA structures in CHIKV and ZIKV
Emerging arboviruses such as Zika virus (ZIKV) and Chikungunya virus (CHIKV) represent a significant threat to human health and have a high potential to cause outbreaks in the near future. At present, there are no specific antivirals available for either of these important pathogens, despite the wide global prevalence of their vector, Aedes spp. mosquitos.
The positive-strand genomes of ZIKV and CHIKV, members of the flavivirus and alphavirus genera respectively, contain functional, structured cis-acting RNA elements which are essential for virus replication. By specifically targeting such RNA elements using antisense locked nucleic acid oligonucleotides (antisense-LNA), we aim to disrupt their function, and analyse the effect this has on virus replication at different life cycle stages.
Obtaining high quality, single-nucleotide-resolution structural data is essential prior to targeting RNA structures. Consequently, we mapped RNA structural elements within the ZIKV 5’ genome region using a combination of biochemical SHAPE probing, thermodynamic models and phylogenetic analysis. We are currently validating our structural data by analysis of mutant phenotypes in a reverse genetic system.
We demonstrate that functional RNA elements in CHIKV can be specifically targeted - inhibiting replication in both sub-genomic replicon and infectious virus systems. Surface plasmon resonance confirmed that an antisense-LNA binds to a specific stem-loop target with a Kd of 310nM and has an IC50 of 35nM in a sub-genomic replicon system. In future work, we aim to investigate selection of RNA-aptamers against CHIKV and target ZIKV genomic stem-loops using antisense-LNAs.
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Interrogation of the CHIKV nsP-host interactome in human and mosquito cells
Chikungunya virus is a human pathogen transmitted by mosquitos. After a genetic adaptation of the virus that allows increased spreading by Aedes albopictus mosquitos, the past decade, Chikungunya virus has spread across the globe. In a significant number of patients, Chikungunya virus causes a chronic, debilitating, arthritic joint pain.
Chikungunya virus replicates in cells of both its vertebrate host and insect vector. To identify cellular pathways that the virus engages to allow optimal replication in these evolutionary distinct organisms we performed AP-MS to identify interaction partners of the viral non-structural proteins (nsPs) in both human and mosquito cells. Mass spectrometric analysis of on-bead-digestions of affinity purifications coupled to MiST analysis allowed sensitive and reproducible identification of a significant number of cellular protein interaction partners of nsP1, -3 and -4. The retrieval of well-established nsP3 interactors, G3BP and Bin1, in both human and mosquito cells validated our approach. Separate nsPs were associated with both shared and unique interaction partners, the latter belonging to different cellular pathways. Comparison of high-confidence interactors of nsP3 in human and mosquito cells identified 25 proteins that associate with nsP3 in both organisms. Functional classification of these shared nsP3 interactors using GO annotation showed engagement of cell-cell adhesion-, Hippo signaling-, ribosomal function- and innate immune signaling pathways by nsP3 in both human and mosquito cells. Interaction of members of each functional group with nsP3 were validated in AP-WB experiments. Functional roles in viral replication of several of these interactors are evaluated using knockouts.
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Interrogation of the CHIKV nsP-host interactome in human and mosquito cells
Chikungunya virus is a human pathogen transmitted by mosquitos. After a genetic adaptation of the virus that allows increased spreading by Aedes albopictus mosquitos, the past decade, Chikungunya virus has spread across the globe. In a significant number of patients, Chikungunya virus causes a chronic, debilitating, arthritic joint pain.
Chikungunya virus replicates in cells of both its vertebrate host and insect vector. To identify cellular pathways that the virus engages to allow optimal replication in these evolutionary distinct organisms we performed AP-MS to identify interaction partners of the viral non-structural proteins (nsPs) in both human and mosquito cells. Mass spectrometric analysis of on-bead-digestions of affinity purifications coupled to MiST analysis allowed sensitive and reproducible identification of a significant number of cellular protein interaction partners of nsP1, -3 and -4. The retrieval of well-established nsP3 interactors, G3BP and Bin1, in both human and mosquito cells validated our approach. Separate nsPs were associated with both shared and unique interaction partners, the latter belonging to different cellular pathways. Comparison of high-confidence interactors of nsP3 in human and mosquito cells identified 25 proteins that associate with nsP3 in both organisms. Functional classification of these shared nsP3 interactors using GO annotation showed engagement of cell-cell adhesion-, Hippo signaling-, ribosomal function- and innate immune signaling pathways by nsP3 in both human and mosquito cells. Interaction of members of each functional group with nsP3 were validated in AP-WB experiments. Functional roles in viral replication of several of these interactors are evaluated using knockouts.
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Development of reverse genetics for Toscana virus
Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, found in the countries surrounding the Mediterranean. It is unusual within the Phlebovirus genus for exhibiting tropism for the central nervous system: TOSV is one of the top causes of seasonal acute meningitis/encephalitis within its range. However, little progress has been made in the study of TOSV, largely due to the lack of a reliable reverse genetics system. We used RNA sequencing to determine the sequence of Toscana strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007). This data was used to construct cDNA plasmids encoding the viral L, M and S antigenomic sequences under control of the T7 RNA promoter to recover recombinant viruses when expressed in BSR-T7/5 CL21 cells. By sequencing amplified viral cDNA, we identified two single base pair mismatches in the original TOSV reference genome (NC_006318, 19, 20), which when corrected restored functionality to the polymerase. We were then able to recover infectious recombinant TOSV (rTOSV) from cDNA clones, as well as establishing a minigenome system. Using reverse genetics, it has been possible to produce the non-structural gene (NSs) deletion mutants (a known interferon antagonist). These strains are in the process of characterisation and are to be used in infection studies in Phlebotomine flies. TOSV vaccine development has been severely hindered by the failure of previous efforts to develop reverse genetics systems for this virus. With a system now in place, it will hopefully be possible to design novel attenuated vaccine candidates.
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Development of reverse genetics for Toscana virus
Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, found in the countries surrounding the Mediterranean. It is unusual within the Phlebovirus genus for exhibiting tropism for the central nervous system: TOSV is one of the top causes of seasonal acute meningitis/encephalitis within its range. However, little progress has been made in the study of TOSV, largely due to the lack of a reliable reverse genetics system. We used RNA sequencing to determine the sequence of Toscana strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007). This data was used to construct cDNA plasmids encoding the viral L, M and S antigenomic sequences under control of the T7 RNA promoter to recover recombinant viruses when expressed in BSR-T7/5 CL21 cells. By sequencing amplified viral cDNA, we identified two single base pair mismatches in the original TOSV reference genome (NC_006318, 19, 20), which when corrected restored functionality to the polymerase. We were then able to recover infectious recombinant TOSV (rTOSV) from cDNA clones, as well as establishing a minigenome system. Using reverse genetics, it has been possible to produce the non-structural gene (NSs) deletion mutants (a known interferon antagonist). These strains are in the process of characterisation and are to be used in infection studies in Phlebotomine flies. TOSV vaccine development has been severely hindered by the failure of previous efforts to develop reverse genetics systems for this virus. With a system now in place, it will hopefully be possible to design novel attenuated vaccine candidates.
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The Basigin (CD147)-CD98 protein complex is involved in Chikungunya virus attachment and entry in human cells
Chikungunya virus (CHIKV), a positive stranded RNA alphavirus, recently reemerged causing multiple outbreaks around the world. Generally, alphaviruses enter the cell via clathrin-mediated endocytosis. Entry is supported by the structural envelope proteins E2 and E1. Different experimental approaches have been applied previously to identify receptor(s) molecules responsible for CHIKV binding and entry in human and mosquito cells. However they cannot account for all CHIKV entry events in all susceptible cell types.
We performed affinity purification coupled to mass spectrometry to identify entry factors of CHIKV in both human and mosquito cells. We transiently expressed the N-terminally Strep-tagged, full-length envelope gene in 293T and C6/36 cells. Affinity purifications were digested on-bead and analyzed by mass spectrometry. MiST analysis allowed the identification of 39 human proteins with a confidence score above 0.8. Twelve proteins were selected for validation with CRISPR/Cas9 knock-out cells. Three separate AP-MS experiments in C6/36 cells led to the identification of 31, 58 and 11 proteins with a MiST score higher than 0.7, of which 19 proteins were chosen for further analysis.
Using knock-out experiments in 293T cells and a reporter CHIKV (ECSA strain) resulted in the identification of the CD147-CD98 protein complex on human cells as possible entry factor. Repetition of this knock-out experiment using an Asian CHIKV strain combined with E2 staining, confirmed these results. CD147 contains 2 immunoglobulin domains which is similar to MXRA8, a previously identified alphavirus entry factor. The interaction of CD147 with E2 was validated on Western Blot after affinity purification.
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Mosquito vector factors driving arbovirus infection in Anopheles
Anopheles mosquitoes are efficient vectors of human malaria, but they are the primary vector of just one arbovirus, O’nyong nyong virus (ONNV). Consequently Anopheles-virus interactions have been relatively unexamined. It is puzzling that almost all arbovirus transmission is mediated by Aedes and its relatives while Anopheles transmit just a single virus. One hypothesis is that antiviral mechanisms are more efficient in Anopheles than Aedes, but ONNV can circumvent them. Here, we combined empirical data and bioinformatic analysis to generate a high-value set of Anopheles candidate genes likely involved in host-virus interactions. Many of the candidate genes are unannotated. We screened our panel of genes through in vitro gene silencing and identified both proviral and antiviral factors in Anopheles mosquitos. So far, the many genes tested have shown an effect on ONNV replication. For instance, the silencing of the unannotated gene AGAP00570 limited 80% of viral replication suggesting its proviral role in the ONNV-Anopheles cells system. Similarly, silencing of the Leucine-Rich repeat IMmune 4 (LRIM4) reduced the viral replication in 78%. On the contrary, preliminary data in another leucine-rich repeat protein, the APL1C, indicates the antiviral activity of this gene in Anopheles cells. ONNV is an emergent virus in Africa with epidemic potential, and these results reveal the host vector factors that influence mosquito-ONNV infection.
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Mosquito vector factors driving arbovirus infection in Anopheles
Anopheles mosquitoes are efficient vectors of human malaria, but they are the primary vector of just one arbovirus, O’nyong nyong virus (ONNV). Consequently Anopheles-virus interactions have been relatively unexamined. It is puzzling that almost all arbovirus transmission is mediated by Aedes and its relatives while Anopheles transmit just a single virus. One hypothesis is that antiviral mechanisms are more efficient in Anopheles than Aedes, but ONNV can circumvent them. Here, we combined empirical data and bioinformatic analysis to generate a high-value set of Anopheles candidate genes likely involved in host-virus interactions. Many of the candidate genes are unannotated. We screened our panel of genes through in vitro gene silencing and identified both proviral and antiviral factors in Anopheles mosquitos. So far, the many genes tested have shown an effect on ONNV replication. For instance, the silencing of the unannotated gene AGAP00570 limited 80% of viral replication suggesting its proviral role in the ONNV-Anopheles cells system. Similarly, silencing of the Leucine-Rich repeat IMmune 4 (LRIM4) reduced the viral replication in 78%. On the contrary, preliminary data in another leucine-rich repeat protein, the APL1C, indicates the antiviral activity of this gene in Anopheles cells. ONNV is an emergent virus in Africa with epidemic potential, and these results reveal the host vector factors that influence mosquito-ONNV infection.
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Louping Ill Virus Outbreak in North-East Wales
The disease louping ill is an encephalitic viral infection that is fatal in sheep (Oves aries) and grouse (Lagopus lagopus). The disease is caused by the louping ill virus (LIV) and transmitted by sheep ticks (Ixodes ricinus) in upland areas of the United Kingdom. Reported outbreaks are sporadic and prevention is mainly targeted at controlling the vector. Despite the implications for animal welfare and the rural economy, research on the epidemiology and control of LIV has been neglected in recent years. In April of 2019, a group of 200 yearling ewes were moved onto hill grazing at a farm near Oswestry on the English/Welsh border. Within 2-3 weeks, ten had developed clinical signs suggestive of neurological impairment, including torticollis, fitting, head shaking and recumbency. A number of the affected ewes were later found dead. Following post mortem, histopathological investigation of brain tissue from an affected ewe, which had undergone euthanasia, detected glial nodules and perivascular cuffing indicating subacute non-suppurative encephalitis. The haemagglutination inhibition serological test was positive and provided evidence for infection with LIV. Reverse transcription polymerase chain reaction (RT-PCR) gave a positive result for samples of the hind brain and the resulting sequence confirmed the presence of LIV. Phylogenetic analysis indicated that the virus showed the highest identity (>99%) with LIV sequences from Aberystwyth in west Wales and was distinct from other LIV isolates found in the north of England. This case study highlights the ongoing threat to UK sheep from LIV.
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Louping Ill Virus Outbreak in North-East Wales
The disease louping ill is an encephalitic viral infection that is fatal in sheep (Oves aries) and grouse (Lagopus lagopus). The disease is caused by the louping ill virus (LIV) and transmitted by sheep ticks (Ixodes ricinus) in upland areas of the United Kingdom. Reported outbreaks are sporadic and prevention is mainly targeted at controlling the vector. Despite the implications for animal welfare and the rural economy, research on the epidemiology and control of LIV has been neglected in recent years. In April of 2019, a group of 200 yearling ewes were moved onto hill grazing at a farm near Oswestry on the English/Welsh border. Within 2-3 weeks, ten had developed clinical signs suggestive of neurological impairment, including torticollis, fitting, head shaking and recumbency. A number of the affected ewes were later found dead. Following post mortem, histopathological investigation of brain tissue from an affected ewe, which had undergone euthanasia, detected glial nodules and perivascular cuffing indicating subacute non-suppurative encephalitis. The haemagglutination inhibition serological test was positive and provided evidence for infection with LIV. Reverse transcription polymerase chain reaction (RT-PCR) gave a positive result for samples of the hind brain and the resulting sequence confirmed the presence of LIV. Phylogenetic analysis indicated that the virus showed the highest identity (>99%) with LIV sequences from Aberystwyth in west Wales and was distinct from other LIV isolates found in the north of England. This case study highlights the ongoing threat to UK sheep from LIV.
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Prevalence and resistance pattern of uropathogens from community settings of different regions: an experience from India
Sarita Mohapatra, Rajashree Panigrahy, Vibhor Tak, Shwetha J. V., Sneha K. C., Susmita Chaudhuri, Swati Pundir, Deepak Kocher, Hitender Gautam, Seema Sood, Bimal Kumar Das, Arti Kapil, Pankaj Hari, Arvind Kumar, Rajesh Kumari, Mani Kalaivani, Ambica R., Harshal Ramesh Salve, Sumit Malhotra and Shashi Kant
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