- 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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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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Identification of candidate molecular determinants of the vector competence of Ixodes ricinus for members of the tick-borne encephalitis complex
In Europe, tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are two flaviviruses both transmitted by the tick Ixodes ricinus. While the mechanisms of viral replication and transmission in this vector are incompletely understood, they are presumed to be largely governed by protein-protein interactions established between viruses and cells.
To elucidate the molecular determinants involved in vector competence, we have mapped the network of protein-protein interactions established between viral proteins of both TBEV and LIV and tick proteins encoded by a cDNA library of I. ricinus, by using yeast two-hybrid methodology. Twenty-two cellular partners from I. ricinus have been identified and all evidenced to interact with both viruses. Upon functional annotation, some of these tick proteins seem to be involved in such biological processes as the immune response or ribosomal maturation. To gain insight into the role of each tick protein in viral replication, the impact of gene silencing will be assessed by dsRNA knockdown. In parallel, I. ricinus cell lines have been persistently infected with TBEV or LIV and the level of expression of selected antiviral effectors monitored over time, in an effort to characterize the antiviral response of this arthropod.
This work represents the first description of the protein-protein interaction network for TBEV, LIV and I. ricinus. Certain tick partners may well represent molecular determinants of vector competence of I. ricinus for TBEV and LIV and potentially other flaviviruses, which we will ascertain by in vivo silencing of selected tick partners.
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Identification of candidate molecular determinants of the vector competence of Ixodes ricinus for members of the tick-borne encephalitis complex
In Europe, tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are two flaviviruses both transmitted by the tick Ixodes ricinus. While the mechanisms of viral replication and transmission in this vector are incompletely understood, they are presumed to be largely governed by protein-protein interactions established between viruses and cells.
To elucidate the molecular determinants involved in vector competence, we have mapped the network of protein-protein interactions established between viral proteins of both TBEV and LIV and tick proteins encoded by a cDNA library of I. ricinus, by using yeast two-hybrid methodology. Twenty-two cellular partners from I. ricinus have been identified and all evidenced to interact with both viruses. Upon functional annotation, some of these tick proteins seem to be involved in such biological processes as the immune response or ribosomal maturation. To gain insight into the role of each tick protein in viral replication, the impact of gene silencing will be assessed by dsRNA knockdown. In parallel, I. ricinus cell lines have been persistently infected with TBEV or LIV and the level of expression of selected antiviral effectors monitored over time, in an effort to characterize the antiviral response of this arthropod.
This work represents the first description of the protein-protein interaction network for TBEV, LIV and I. ricinus. Certain tick partners may well represent molecular determinants of vector competence of I. ricinus for TBEV and LIV and potentially other flaviviruses, which we will ascertain by in vivo silencing of selected tick partners.
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Monitoring of Insecticide Resistance on Aedes sp. Mosquitoes in Banyumas Regency, Indonesia
Background. The continuous use of insecticides for mosquito-borne disease control can cause insecticide resistance, and if left unchecked, this could lead to a substantial increase in disease incidence. The aims of this study are to monitor insecticide insecticides against Malathion and Cypermethrin and identifying the mechanisms underlying the resistance in the area of study.
Method. This is a descriptive study located in Banyumas Regency. Aedes sp. mosquitoes werecollected from three endemic areas (Arcawinangun, Karangpucung, and Purwanegara) by the ovitrap installation to 100 houses each village (total 300 houses). Filial 1 of Aedes were tested their insecticide resistance to Malathion and Cypermethrin by susceptibility test, biochemical assay and molecular by PCR.
Results. The results of the susceptibility test showed the average percentage of mosquito mortality from three villages was 30,67% which were included in the resistance category. However, the results of the biochemical assay showed that 70% of mosquitoes are still very susceptible (AV<0,7). Molecular tests are underway and the results are likely to be obtained in August 2019
Conclusion. The population of Aedes sp. in the study area has been resistant to malathion and cypermethrin, and the mechanism underlying this resistance was not based on a biochemical mechanism. It is necessary to rotate the use of insecticide active substances in DHF vector control by selecting insecticides that have a different mode of action.
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Monitoring of Insecticide Resistance on Aedes sp. Mosquitoes in Banyumas Regency, Indonesia
Background. The continuous use of insecticides for mosquito-borne disease control can cause insecticide resistance, and if left unchecked, this could lead to a substantial increase in disease incidence. The aims of this study are to monitor insecticide insecticides against Malathion and Cypermethrin and identifying the mechanisms underlying the resistance in the area of study.
Method. This is a descriptive study located in Banyumas Regency. Aedes sp. mosquitoes werecollected from three endemic areas (Arcawinangun, Karangpucung, and Purwanegara) by the ovitrap installation to 100 houses each village (total 300 houses). Filial 1 of Aedes were tested their insecticide resistance to Malathion and Cypermethrin by susceptibility test, biochemical assay and molecular by PCR.
Results. The results of the susceptibility test showed the average percentage of mosquito mortality from three villages was 30,67% which were included in the resistance category. However, the results of the biochemical assay showed that 70% of mosquitoes are still very susceptible (AV<0,7). Molecular tests are underway and the results are likely to be obtained in August 2019
Conclusion. The population of Aedes sp. in the study area has been resistant to malathion and cypermethrin, and the mechanism underlying this resistance was not based on a biochemical mechanism. It is necessary to rotate the use of insecticide active substances in DHF vector control by selecting insecticides that have a different mode of action.
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Salivary gland RNA-seq from arbovirus-infected Aedes aegypti and Aedes albopictus provides insights into virus transmission
Yellow fever mosquitoes (Aedes aegypti) and Asian tiger mosquitoes (Aedes albopictus) are the primary vectors of dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV). These viruses are transmitted to humans through mosquito saliva, making the vector salivary gland (SG) a critical tissue to identify transmission-blocking targets. We examined gene expression in infected SGs for both vector species and for three different virus infections. Aedes aegypti SGs were infected separately with DENV, ZIKV and CHIKV, and Ae. albopictus with CHIKV. RNA-sequencing identified differentially expressed coding and long non-coding RNAs (lncRNAs). Differentially expressed genes determined from genome annotations were greater in number and functional diversity in comparison to differentially expressed transcripts from de novo transcriptome assemblies. Salivary protein transcripts were the most abundant, but were downregulated in all three virus infections. Commonly upregulated genes were associated with apoptosis, cytoskeletal proteins, replication/transcription/translation, redox/stress and immunity. An enrichment of upregulated genes related to apoptosis were observed in CHIKV infection in comparison to DENV and ZIKV infections. Upregulation of serine proteases and other genes associated with immunity and cellular stress responses (cytochrome P450 genes) varied between vectors. There were also immune response commonalities between vectors, for instance RNA-interference was observed to be a non-specific antiviral defense. The number of lncRNA transcripts differentially expressed were few and none were common to all infections, likely having minor roles, unlike the lncRNA antiviral effects proposed for mosquito midgut. Determining common infection patterns for different viruses and vectors has applications in refractory vector engineering.
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Salivary gland RNA-seq from arbovirus-infected Aedes aegypti and Aedes albopictus provides insights into virus transmission
Yellow fever mosquitoes (Aedes aegypti) and Asian tiger mosquitoes (Aedes albopictus) are the primary vectors of dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV). These viruses are transmitted to humans through mosquito saliva, making the vector salivary gland (SG) a critical tissue to identify transmission-blocking targets. We examined gene expression in infected SGs for both vector species and for three different virus infections. Aedes aegypti SGs were infected separately with DENV, ZIKV and CHIKV, and Ae. albopictus with CHIKV. RNA-sequencing identified differentially expressed coding and long non-coding RNAs (lncRNAs). Differentially expressed genes determined from genome annotations were greater in number and functional diversity in comparison to differentially expressed transcripts from de novo transcriptome assemblies. Salivary protein transcripts were the most abundant, but were downregulated in all three virus infections. Commonly upregulated genes were associated with apoptosis, cytoskeletal proteins, replication/transcription/translation, redox/stress and immunity. An enrichment of upregulated genes related to apoptosis were observed in CHIKV infection in comparison to DENV and ZIKV infections. Upregulation of serine proteases and other genes associated with immunity and cellular stress responses (cytochrome P450 genes) varied between vectors. There were also immune response commonalities between vectors, for instance RNA-interference was observed to be a non-specific antiviral defense. The number of lncRNA transcripts differentially expressed were few and none were common to all infections, likely having minor roles, unlike the lncRNA antiviral effects proposed for mosquito midgut. Determining common infection patterns for different viruses and vectors has applications in refractory vector engineering.
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Transcriptomic analysis of human neurons and astrocytes infected with TBEV strains of different virulence
Tick-borne encephalitis virus (TBEV; Flaviviridae) can cause serious infections in humans which may result in encephalitis/meningoencephalitis. It has been previously reported that TBEV infects both, neurons and astrocytes, however, with a different outcome. So far, the principle of this cell type-specific response to TBEV is not fully understood.
In order to gain more insight into this phenomenon, we described new in vitro infection model utilizing human neural stem cells (hNSCs) and two strains of Western European TBEV subtype varying in the pathogenicity - mild Neudoerfl and severe Hypr. In detail, neurons and astrocytes were artificially differentiated from hNSCs and presence of CNS markers was checked. TBEV infection in both cell types was characterised afterwards. As expected, both cell types proved to be susceptible to TBEV infection. Viability was negatively affected only in infected neurons. In order to identify possible effectors responsible for different susceptibility of neurons and astrocytes, the analyses of changes in poly-(A) and small RNA transcriptome upon TBEV infection were performed. Preliminary results from poly-(A) RNA transcriptome revealed that in both cell types mainly interferon-stimulated genes (ISGs) were up-regulated. However, the expression kinetics of particular ISGs varied. In addition, the vast spectrum of long non-coding RNAs was described to be differentially expressed upon infection. Surprisingly, U1 snRNA was found to be the most down-regulated RNA species among almost all infected samples.
Further analyses are in progress in order to get a complete description of virus-induced changes on the transcriptomic level.
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Transcriptomic analysis of human neurons and astrocytes infected with TBEV strains of different virulence
Tick-borne encephalitis virus (TBEV; Flaviviridae) can cause serious infections in humans which may result in encephalitis/meningoencephalitis. It has been previously reported that TBEV infects both, neurons and astrocytes, however, with a different outcome. So far, the principle of this cell type-specific response to TBEV is not fully understood.
In order to gain more insight into this phenomenon, we described new in vitro infection model utilizing human neural stem cells (hNSCs) and two strains of Western European TBEV subtype varying in the pathogenicity - mild Neudoerfl and severe Hypr. In detail, neurons and astrocytes were artificially differentiated from hNSCs and presence of CNS markers was checked. TBEV infection in both cell types was characterised afterwards. As expected, both cell types proved to be susceptible to TBEV infection. Viability was negatively affected only in infected neurons. In order to identify possible effectors responsible for different susceptibility of neurons and astrocytes, the analyses of changes in poly-(A) and small RNA transcriptome upon TBEV infection were performed. Preliminary results from poly-(A) RNA transcriptome revealed that in both cell types mainly interferon-stimulated genes (ISGs) were up-regulated. However, the expression kinetics of particular ISGs varied. In addition, the vast spectrum of long non-coding RNAs was described to be differentially expressed upon infection. Surprisingly, U1 snRNA was found to be the most down-regulated RNA species among almost all infected samples.
Further analyses are in progress in order to get a complete description of virus-induced changes on the transcriptomic level.
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Transcriptional and translation shut-off in TBEV infected neural cells and involvement of viral C protein
Tick-borne encephalitis virus (TBEV, Flaviviridae) infection causes severe neurological disease and incapacitates more than 10 000 patients annually in the Eurasian region. Despite extensive studies, some areas of interaction of TBEV with the host cells remain undescribed. Here we investigated the interaction of TBEV and human neural DAOY HTB-186 cells on the transcriptional and translational level.
By labelling of nascent RNA and protein molecules in TBEV-infected DAOY cells, we showed that the virus-induced host translational shut-off. Moreover, TBEV interfered also with the expression of host ribosomal RNAs, in particular with the rRNA species transcribed by RNA polymerase I (18S rRNA, 28S rRNA, and their precursor 45-47S pre-rRNA). Synthesis of host rRNAs is an essential host cell process that is localized in the nucleus, namely nucleoli. By searching for virus factor that could be linked with these effects, we described so far unknown nucleolar localization of TBEV capsid protein C. More importantly, preliminary data from transfection of recombinant C protein led to the reduction in nascent protein synthesis indicating the link between TBEV capsid protein and shut-off phenomena which were described. Furthermore, we identified a potential nuclear localization signal, which seems not to be essential for the shut-down effect.
Taken together we described a brand new type of interaction between TBEV and host neural cells on the transcriptional and translational level and identified viral factor potentially responsible for the observed phenomena. However, further analyses are needed, and the particular mechanism of action remains still elusive.
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Transcriptional and translation shut-off in TBEV infected neural cells and involvement of viral C protein
Tick-borne encephalitis virus (TBEV, Flaviviridae) infection causes severe neurological disease and incapacitates more than 10 000 patients annually in the Eurasian region. Despite extensive studies, some areas of interaction of TBEV with the host cells remain undescribed. Here we investigated the interaction of TBEV and human neural DAOY HTB-186 cells on the transcriptional and translational level.
By labelling of nascent RNA and protein molecules in TBEV-infected DAOY cells, we showed that the virus-induced host translational shut-off. Moreover, TBEV interfered also with the expression of host ribosomal RNAs, in particular with the rRNA species transcribed by RNA polymerase I (18S rRNA, 28S rRNA, and their precursor 45-47S pre-rRNA). Synthesis of host rRNAs is an essential host cell process that is localized in the nucleus, namely nucleoli. By searching for virus factor that could be linked with these effects, we described so far unknown nucleolar localization of TBEV capsid protein C. More importantly, preliminary data from transfection of recombinant C protein led to the reduction in nascent protein synthesis indicating the link between TBEV capsid protein and shut-off phenomena which were described. Furthermore, we identified a potential nuclear localization signal, which seems not to be essential for the shut-down effect.
Taken together we described a brand new type of interaction between TBEV and host neural cells on the transcriptional and translational level and identified viral factor potentially responsible for the observed phenomena. However, further analyses are needed, and the particular mechanism of action remains still elusive.
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The Using of Serum Free Media for the Production of J93-463-1-16-10 Monoclonal Antibody against Japanese encephalitis virus
More LessThe development of J93-463-1-16-10 cell culture by sequential adaptation method in serum-free media of Hybridoma-SFM medium and CD Hybridoma medium to produce the monoclonal antibody specific to Japanese encephalitis virus was shown that the viabilities of J93-463-1-16-10 were more than 70%. Then the supernatant from the cell culture was purified after precipitation with ammonium sulfate until the final concentration was 50% in order to separate the IgG. After that, the protein concentrations were measured by using the Bradford assay. Next, the proteins were separated, which made each protein purer with SDS-PAGE method in order to find the molecular weights of the separated proteins. The putative IgG and its heavy chains and light chains were divided in two sizes which were approximate 23-25 kDa and 50-53 kDa respectively. Later, the type of immunoglobulin will be identified by the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) method and its antibody characteristic to Japanese encephalitis virus which will be done further.
In the future, a monoclonal antibody in this research could be used to study the expression of the envelope protein of Japanese encephalitis virus in green cos and red cos, and to develop the vaccine for Japanese encephalitis virus in swine.
Keywords: Japanese encephalitis virus, J93-463-1-16-10, serum-free media, MALDI-TOF
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The Cryopreservation of Cos Lettuce Callus by Using Liquid Nitrogen
More LessThe cryopreservation of cos lettuce (CL) callus by using liquid nitrogen (LN) was further study from the previous research on callus induction and micropropagation of CL. That was studied on seed culture and micropropagation of CL and its embryogenic growth. The result was shown that the most suitable Murashing and Skoog (MS) medium concentration was in 1/2 MS. The medium supplemented with 0.1 mg/l of 6-benzylaminopurine (BAP) and 0.5 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D) gave the best for leaf inducing callus. Then it was brought to study the effects of cryoprotectants with the different percentages of dimethyl sulfoxide (DMSO). The cryopreserved callus by using LN was brought to subculture for the best inducing shoot in MS medium supplemented with 4.0 mg/l of BAP. The results that the best cryoprotectant at the percentage of DMSO at 7.5 gave the highest average number of shoots at 2.1 shoots/culture and the average shoot length was 1.56 cm. The MS medium supplemented with 0.5 mg/l of 1-Naphthaleneacetic acid was the best inducing root. The results that the best cryoprotectant at the percentage of DMSO at 7.5 gave the highest average number of roots at 9.7 roots/culture and the average root length was 1.59 cm. Plant differentiation from callus of CL leaf was transferred to grow in soil and had the same morphology as normal CL that was grown in soil by seed germination.
CL will be the host of Japanese encephalitis virus expression for the developmental swine vaccine.
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Toolkit for the study of yellow fever virus
Yellow fever virus (YFV) is a dangerous re-emerging mosquito borne flavivirus with a high lethality, which causes untreatable haemorrhagic fever in humans. Important disadvantages regarding safety and limited vaccine supply have been identified in the existing live attenuated vaccine. Alternative safer therapeutic strategies are needed to reduce the lethality of infections and protect a wider group of people. To this point, we created an array of essential tools for the study of YFV. We designed a novel YFV reporter virus to develop a highly sensitive high throughput neutralisation assay (Z’=0.65), which can screen virus inhibitors in only 48 hours. Moreover, we designed a site-specific biotinylated soluble YF envelope (E) protein by a cloning strategy involving the use of a biotin acceptor peptide and E. coli Biotin-protein birA ligase. The biotinylated E protein was used to develop a highly sensitive ELISA to screen antibodies that bind to the YFV E protein. Furthermore, we immunised mice with a soluble YFV E protein and generated anti-YFV E IgG secreting hybridoma cell lines. Monoclonal antibodies secreted by hybridomas were purified by affinity chromatography and are being characterised. The developed antibodies against YF E protein are a crucial tool for the molecular study of YF. Additionally, neutralising antibodies against YFV could potentially be developed into the first therapeutic treatment against YF infection. The toolkit developed in this project includes a neutralisation assay, a binding ELISA, and anti YFV E antibodies. These are essential instruments to expand the knowledge of YFV.
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Toolkit for the study of yellow fever virus
Yellow fever virus (YFV) is a dangerous re-emerging mosquito borne flavivirus with a high lethality, which causes untreatable haemorrhagic fever in humans. Important disadvantages regarding safety and limited vaccine supply have been identified in the existing live attenuated vaccine. Alternative safer therapeutic strategies are needed to reduce the lethality of infections and protect a wider group of people. To this point, we created an array of essential tools for the study of YFV. We designed a novel YFV reporter virus to develop a highly sensitive high throughput neutralisation assay (Z’=0.65), which can screen virus inhibitors in only 48 hours. Moreover, we designed a site-specific biotinylated soluble YF envelope (E) protein by a cloning strategy involving the use of a biotin acceptor peptide and E. coli Biotin-protein birA ligase. The biotinylated E protein was used to develop a highly sensitive ELISA to screen antibodies that bind to the YFV E protein. Furthermore, we immunised mice with a soluble YFV E protein and generated anti-YFV E IgG secreting hybridoma cell lines. Monoclonal antibodies secreted by hybridomas were purified by affinity chromatography and are being characterised. The developed antibodies against YF E protein are a crucial tool for the molecular study of YF. Additionally, neutralising antibodies against YFV could potentially be developed into the first therapeutic treatment against YF infection. The toolkit developed in this project includes a neutralisation assay, a binding ELISA, and anti YFV E antibodies. These are essential instruments to expand the knowledge of YFV.
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The Tick Cell Biobank: new arthropod cell lines for arbovirus research
More LessThe Tick Cell Biobank is the world’s only dedicated culture collection for cell lines derived from ticks and other arthropod vectors. As well as storing and distributing arthropod cell lines and training in their maintenance to UK and international researchers, the Tick Cell Biobank generates novel cell lines from arthropod species and geographic strains not already represented in the collection. Currently, efforts are focussed on European Argas, Dermacentor, Hyalomma, Rhipicephalus and Ixodes spp. ticks, Lutzomyia and Phlebotomus spp. sandflies, Culicoides spp. biting midges, Rhodnius prolixus kissing bugs and Glossina morsitans tsetse flies. Techniques used previously for ticks and insects are applied or adapted for use with embryonic or larval arthropods to generate primary cell and tissue cultures; these primary cultures are then maintained until significant cell multiplication commences and subculture can be attempted, which may take several years. This approach has yielded new cell lines from the soft tick Argas reflexus, the hard tick Hyalomma lusitanicum, the New World sand fly Lutzomyia longipalpis and the UK midge Culicoides nubeculosus. Most of these novel cell lines are now available for arbovirus research through the Tick Cell Biobank.
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AurKB activity is necessary for Dengue virus release
Flaviviruses, such as Dengue (DENV), Zika and Yellow Fever are pathogens with high morbidity and mortality. Around 390 million people per year are infected with DENV, and almost 90 million develop the clinical forms of the infection. In the present work, we analyzed the role of Aurora Kinase B (AurKB) in the replicative cycle of DENV. This Kinase regulates the activation of ESCRT-III complex, which has an essential role in the viral morphogenesis and/or budding from RE to Golgi apparatus. The compound ZM 447439 (ZM) was used to inhibit specifically AurKB, and the viral progeny, viral RNA/protein synthesis efficiency and NS1 secretion were evaluated. The kinase inhibition did not alter the viral protein production/secretion or genome replication but impaired the viral yield without altering the percentage of infected cells.
Moreover, confocal microscopy analysis of DENV-infected ZM447439-treated cells shows a delocalization of viral components from the replicative complexes. In summary, these observations indicate that AurKB participates in DENV viral morphogenesis or release. Together, our results suggest possible participation of AurKB in the viral release of budding through activation of the ESCRT-III complex and suggest a new role for AurKB on flavivirus viral cycle.
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AurKB activity is necessary for Dengue virus release
Flaviviruses, such as Dengue (DENV), Zika and Yellow Fever are pathogens with high morbidity and mortality. Around 390 million people per year are infected with DENV, and almost 90 million develop the clinical forms of the infection. In the present work, we analyzed the role of Aurora Kinase B (AurKB) in the replicative cycle of DENV. This Kinase regulates the activation of ESCRT-III complex, which has an essential role in the viral morphogenesis and/or budding from RE to Golgi apparatus. The compound ZM 447439 (ZM) was used to inhibit specifically AurKB, and the viral progeny, viral RNA/protein synthesis efficiency and NS1 secretion were evaluated. The kinase inhibition did not alter the viral protein production/secretion or genome replication but impaired the viral yield without altering the percentage of infected cells.
Moreover, confocal microscopy analysis of DENV-infected ZM447439-treated cells shows a delocalization of viral components from the replicative complexes. In summary, these observations indicate that AurKB participates in DENV viral morphogenesis or release. Together, our results suggest possible participation of AurKB in the viral release of budding through activation of the ESCRT-III complex and suggest a new role for AurKB on flavivirus viral cycle.
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Implications of vertical transmission of Alphaviruses in Aedes aegypti mosquitoes
Mosquitoes are vectors for epidemic transmission of viruses of public and veterinary health. The mosquito vector is generally infected for life although, unlike the vertebrate counterpart, does not suffer a high fitness cost. Having a vector infected for the length of its life enables routes of viral transmission other than the classic infected bite (horizontal), including sexual (horizontal) and to the progeny (vertical). Vertical transmission is considered a route of transmission that allows for the persistence of the virus during adverse environmental periods (e.g., droughts, cold periods). Because Aedes aegypti and Aedes albopictus eggs are resistant to dessication, it is hypothesised that this attribute could promote arbovirus survival between transmission cycles, playing an important role in maintaining the pathogen.
Vertical transmission of arboviruses has been extensively documented for flaviviruses and bunyaviruses. However, there is very little and contradictory reports of vertical transmission of alphaviruses. In this research we establish the mechanisms of vertical transmission of the alphaviruses Semiliki Forest virus (SFV) and Ross river virus (RRV) and its implications in pathogen transmission of future generations.
Aedes aegypti mosquitoes were infected with SFV virus in different gonotrophic cycles. Their offspring was then reared and challenged with SFV, RRV and dengue virus (DENV). Offspring from infected parents showed significant reduction in viral load if infected with SFV or RRV but not with DENV.
Findings of this research highlight the importance of vertical transmission of alphaviruses in the general arbovirus infectious cycle.
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Implications of vertical transmission of Alphaviruses in Aedes aegypti mosquitoes
Mosquitoes are vectors for epidemic transmission of viruses of public and veterinary health. The mosquito vector is generally infected for life although, unlike the vertebrate counterpart, does not suffer a high fitness cost. Having a vector infected for the length of its life enables routes of viral transmission other than the classic infected bite (horizontal), including sexual (horizontal) and to the progeny (vertical). Vertical transmission is considered a route of transmission that allows for the persistence of the virus during adverse environmental periods (e.g., droughts, cold periods). Because Aedes aegypti and Aedes albopictus eggs are resistant to dessication, it is hypothesised that this attribute could promote arbovirus survival between transmission cycles, playing an important role in maintaining the pathogen.
Vertical transmission of arboviruses has been extensively documented for flaviviruses and bunyaviruses. However, there is very little and contradictory reports of vertical transmission of alphaviruses. In this research we establish the mechanisms of vertical transmission of the alphaviruses Semiliki Forest virus (SFV) and Ross river virus (RRV) and its implications in pathogen transmission of future generations.
Aedes aegypti mosquitoes were infected with SFV virus in different gonotrophic cycles. Their offspring was then reared and challenged with SFV, RRV and dengue virus (DENV). Offspring from infected parents showed significant reduction in viral load if infected with SFV or RRV but not with DENV.
Findings of this research highlight the importance of vertical transmission of alphaviruses in the general arbovirus infectious cycle.
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JNK pathway-a key mediator of antiviral immunity in mosquito salivary glands
Background: Mosquito salivary glands play crucial role in transmission of arboviral diseases like Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV). We aimed to characterize virus responsive gene expression in Aedes aegypti salivary glands against these pathogenic arboviruses.
Methods: We performed high throughput RNA-sequencing on uninfected and virus-infected (DENV, ZIKV, CHIKV) female Ae. aegypti salivary glands to elucidate differential expression of genes at the transcript level. We validated the transcriptomic analysis by qPCR and performed RNA-i based functional characterization of virus-induced immune genes.
Results: DENV, ZIKV or CHIKV infected salivary gland transcriptome revealed regulations of genes related to blood feeding, metabolism, apoptosis, and immunity; the latter including Toll, IMD, and JNK pathway components. Silencing of Toll and IMD pathway components did not increase replication of all three viruses. However, depletion of infection induced JNK pathway activator and repressor showed conserved antiviral response of this pathway against the viral infections. We further showed that JNK activation by arboviruses is mediated by antiviral complement and apoptosis activation.
Conclusion: This study determined the previously unknown antiviral mechanism of JNK pathway in mosquito salivary glands against important pathogenic arboviruses. This pathway shows potential to be utilized for developing effective transmission blocking tools.
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JNK pathway-a key mediator of antiviral immunity in mosquito salivary glands
Background: Mosquito salivary glands play crucial role in transmission of arboviral diseases like Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV). We aimed to characterize virus responsive gene expression in Aedes aegypti salivary glands against these pathogenic arboviruses.
Methods: We performed high throughput RNA-sequencing on uninfected and virus-infected (DENV, ZIKV, CHIKV) female Ae. aegypti salivary glands to elucidate differential expression of genes at the transcript level. We validated the transcriptomic analysis by qPCR and performed RNA-i based functional characterization of virus-induced immune genes.
Results: DENV, ZIKV or CHIKV infected salivary gland transcriptome revealed regulations of genes related to blood feeding, metabolism, apoptosis, and immunity; the latter including Toll, IMD, and JNK pathway components. Silencing of Toll and IMD pathway components did not increase replication of all three viruses. However, depletion of infection induced JNK pathway activator and repressor showed conserved antiviral response of this pathway against the viral infections. We further showed that JNK activation by arboviruses is mediated by antiviral complement and apoptosis activation.
Conclusion: This study determined the previously unknown antiviral mechanism of JNK pathway in mosquito salivary glands against important pathogenic arboviruses. This pathway shows potential to be utilized for developing effective transmission blocking tools.
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The effect of co-infecting Anaplasma phagocytophilum on replication of Langat Virus, a model for Louping ill virus, in Ixodes spp. tick cells
More LessTick borne fever (TBF) caused by Anaplasma phagocytophilum (Ap), and louping ill caused by the flavivirus louping ill virus (LIV) are the two most economically important vector-borne diseases in UK sheep populations. Both pathogens are transmitted by the tick Ixodes ricinus, which also harbours protozoan parasites and Borrelia spp. spirochetes. I. ricinus ticks may be co-infected with multiple microorganisms and potentially transmit more than one pathogen to hosts during blood feeding.
Ap infection is not limited to sheep, causing pasture fever in cattle and granulocytic anaplasmosis in horses, dogs and humans. There is no vaccine available for TBF, and disease control relies on tick control and antibiotic treatment. LIV mainly infects ruminants, but can also infect other livestock including horses, pigs, alpacas and llamas. Since 2016, the LIV vaccine has been unavailable and there is no alternative prophylactic treatment for livestock. Both Ap and LIV are zoonotic diseases with occasional human cases reported in the UK.
Importantly, Ap infection leads to host immunosuppression and consequently increases vulnerability to secondary infections. Both Ap and LIV have been studied as single infections in tick and mammalian cells. However the dynamics and implications of co-infections within the arthropod vector or mammalian host have, to date, not been fully explored.
Using embryo-derived Ixodes spp. cell lines infected with Langat virus (a BSL-2 model for LIV) and Ap, we examined the effect of co-infection on viral RNA replication by qRT-PCR and bacterial growth by qPCR. The results of this study will be presented and discussed.
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The emerged genotype I of Japanese encephalitis virus shows an infectivity similar to genotype III in Culex pipiens mosquitoes
Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that represents the most significant etiology of childhood viral neurological infections throughout the Asia. During the last 20 years, JEV genotype dominance has shifted from genotype III (GIII) to genotype I (GI). To date, the exact mechanism of this displacement is still not known. Culex (Cx.) mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle. In this study, we used Cx. pipiens mosquitoes from China as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift. We exposed female Cx. pipiens mosquitoes orally to either GI or GIII JEV strains. Midgut, whole mosquitoes, secondary organs, and salivary glands of JEV-infected mosquitoes were collected at 7 and 14 days of post infection (dpi) and subjected to measure the infection rate, replication kinetics, dissemination rate and transmission potential of the infected JEV strains in Cx. pipiens mosquitoes by 50% tissue culture infective dose assay. We found that Cx. pipiens mosquito was competent vector for both GI and GIII JEV infection, with similar infection rates and growth kinetics. After the establishment of infection, Cx. pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar rates of dissemination. A few GI-infected mosquito salivary glands (16.2%) were positive for GI virus, whereas GIII virus was undetectable in GIII-infected mosquito salivary glands at 7 dpi. However, 29.4% (5/17) and 36.3% (8/22) were positive for GI- and GIII-infected mosquito salivary glands at 14 dpi, respectively, showing an increase in JEV positive rate. No statistical difference in the transmission rate between GI- and GIII-infected mosquitoes was detected. Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx. pipiens mosquitoes, suggesting that Cx. pipiens mosquitoes from China may not play a critical role in JEV genotype shift. Although the current data were obtained solely from Cx. pipiens mosquitoes, it is likely that the conclusion drawn could be extrapolated to the role of mosquitoes in JEV genotype shift.
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The emerged genotype I of Japanese encephalitis virus shows an infectivity similar to genotype III in Culex pipiens mosquitoes
Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that represents the most significant etiology of childhood viral neurological infections throughout the Asia. During the last 20 years, JEV genotype dominance has shifted from genotype III (GIII) to genotype I (GI). To date, the exact mechanism of this displacement is still not known. Culex (Cx.) mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle. In this study, we used Cx. pipiens mosquitoes from China as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift. We exposed female Cx. pipiens mosquitoes orally to either GI or GIII JEV strains. Midgut, whole mosquitoes, secondary organs, and salivary glands of JEV-infected mosquitoes were collected at 7 and 14 days of post infection (dpi) and subjected to measure the infection rate, replication kinetics, dissemination rate and transmission potential of the infected JEV strains in Cx. pipiens mosquitoes by 50% tissue culture infective dose assay. We found that Cx. pipiens mosquito was competent vector for both GI and GIII JEV infection, with similar infection rates and growth kinetics. After the establishment of infection, Cx. pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar rates of dissemination. A few GI-infected mosquito salivary glands (16.2%) were positive for GI virus, whereas GIII virus was undetectable in GIII-infected mosquito salivary glands at 7 dpi. However, 29.4% (5/17) and 36.3% (8/22) were positive for GI- and GIII-infected mosquito salivary glands at 14 dpi, respectively, showing an increase in JEV positive rate. No statistical difference in the transmission rate between GI- and GIII-infected mosquitoes was detected. Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx. pipiens mosquitoes, suggesting that Cx. pipiens mosquitoes from China may not play a critical role in JEV genotype shift. Although the current data were obtained solely from Cx. pipiens mosquitoes, it is likely that the conclusion drawn could be extrapolated to the role of mosquitoes in JEV genotype shift.
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A Viral Metagenomic Analysis Reveals Rich Viral Abundance and Diversity in Mosquitoes from Pig Farms
Mosquitoes harbor a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns and significant economic losses across the globe. The viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.
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A Viral Metagenomic Analysis Reveals Rich Viral Abundance and Diversity in Mosquitoes from Pig Farms
Mosquitoes harbor a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns and significant economic losses across the globe. The viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.
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The Expression of Japanese Encephalitis Virus Envelope Gene in Green Cos
More LessThe expression of Japanese encephalitis virus (JEV) envelope gene in green cos leaf was a project undertaken at the John Innes Centre during 6 September to 20 October 2018 and is one of the projects of the research training program at the John Innes Centre during long vacation every year since 2010 until now.
The summary of this research is as follows. The production of the envelope protein of the JEV in green cos by using the envelope gene expression by 8 epitopes (MEP: multiepitope) of Japanese encephalitis virus vaccine strain SA14-14-2 that provides the highest immune response against JEV. After incorporated the envelope gene into pEAQ-HT and pEAQ-HT-HBcAg-tEL vectors, we cloned genes in Escherichia coli and Agrobacterium tumefaciens, respectively, then expressed the envelope protein of JEV in green cos leaves. The result can be seen in photographs of green cos leaves by both visible light camera and ultraviolet light camera.
Further research should include the analysis and identification by chemiluminescence immunoassay and by the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). Further purification of the envelope protein expression of JEV in green cos that will be benefited for the production of the envelope protein of the JEV plant-based vaccine.
Key words: Japanese encephalitis virus, plant-based vaccine, chemiluminescence immunoassay, MALDI-TOF
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Zika virus infection of glia leads to secondary injury to axons and dendrites
Zika virus infection was recently linked to microcephaly and peripheral neuropathy (GBS) in Zika virus epidemic areas. Building on our previous work (Cumberworth et al, 2017) we investigated, in a time-course study, how the viral infection and the injury of cell processes of oligodendrocytes and neurons are related to each other in the same in vitro model.
We generated CNS myelinating cultures from a reporter mouse (Thy1-YFP) on the Ifnar1 -/-background. A proportion of neurons and their processes are positive for YFP in those cultures, which enabled us to visualise single neurons. Cultures were infected with the Brazilian Zika virus strain (PE243) at an MOI of 0.3 and cultured for up to 6 days post infection. We observed that the neuronal cell processes were affected as early as the appearance of the first clusters of infected glial cells.
To analyse the interrelation of neurons and myelin further, cultures were labeled with an antibody recognising proteolipid protein. We found that the myelin got injured as early as neuronal processes. These results suggest that the injury to neuronal processes might be a consequence of the infection of the primary target of Zika virus: oligodendroglia.
These data help us to understand disease pathogenesis of Zika virus infection of the CNS, and whether there is a time-window to intervene therapeutically. Furthermore, this gives an insight as to how viral infection of glial cells can affect neuronal processes such as axons.
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Zika virus infection of glia leads to secondary injury to axons and dendrites
Zika virus infection was recently linked to microcephaly and peripheral neuropathy (GBS) in Zika virus epidemic areas. Building on our previous work (Cumberworth et al, 2017) we investigated, in a time-course study, how the viral infection and the injury of cell processes of oligodendrocytes and neurons are related to each other in the same in vitro model.
We generated CNS myelinating cultures from a reporter mouse (Thy1-YFP) on the Ifnar1 -/-background. A proportion of neurons and their processes are positive for YFP in those cultures, which enabled us to visualise single neurons. Cultures were infected with the Brazilian Zika virus strain (PE243) at an MOI of 0.3 and cultured for up to 6 days post infection. We observed that the neuronal cell processes were affected as early as the appearance of the first clusters of infected glial cells.
To analyse the interrelation of neurons and myelin further, cultures were labeled with an antibody recognising proteolipid protein. We found that the myelin got injured as early as neuronal processes. These results suggest that the injury to neuronal processes might be a consequence of the infection of the primary target of Zika virus: oligodendroglia.
These data help us to understand disease pathogenesis of Zika virus infection of the CNS, and whether there is a time-window to intervene therapeutically. Furthermore, this gives an insight as to how viral infection of glial cells can affect neuronal processes such as axons.
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Of Mice and Monkeys: Determining Protective Serological Titres in Model Zika Virus Infections
Developmental vaccines against emerging pathogens face many hurdles including determining what protective level of serological responses must be generated. Knowledge of a likely protective titre is critical where human challenge studies are not possible.
We have used an anti-zika plasma pool from convalescent patients (candidate serological reference reagent NIBSC16/320-14), infused into cynomolgus macaques and Type-1 IFN deficient mice to determine likely protective neutralising titres.
Anti-zika plasma was administered to a group of 4 macaques (single concentration) and groups of 8 A129 mice (4 group titration series) 24 hours prior to sub-cutaneous challenge with Zika virus PRVABC59. Plasma/sera samples were collected at regular intervals to track peripheral viremia and anti-zika antibody responses. FFPE tissues were collected at termination for histological analysis.
Immediately prior to challenge, human IgG was detectable in all infused animals. Within macaques the NT50 at this time was 250. All macaques that received plasma were protected against zika virus infection as determined by plasma/tissue qRT-PCR and IgM responses.
Titration within A129 mice gave a neutralisation titre of 110, above which mice were generally protected against zika infection. However this was not absolute as a small number of mice with high neutralisation levels were infected.
A protective NT50 of 250 has been identified for macaques and this further titrated in A129 mice to give a guide protective neutralisation titre of 110. The lack of protection in some mice with higher titres is currently unclear and studies are underway to compare their infection pathology with that of controls.
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Of Mice and Monkeys: Determining Protective Serological Titres in Model Zika Virus Infections
Developmental vaccines against emerging pathogens face many hurdles including determining what protective level of serological responses must be generated. Knowledge of a likely protective titre is critical where human challenge studies are not possible.
We have used an anti-zika plasma pool from convalescent patients (candidate serological reference reagent NIBSC16/320-14), infused into cynomolgus macaques and Type-1 IFN deficient mice to determine likely protective neutralising titres.
Anti-zika plasma was administered to a group of 4 macaques (single concentration) and groups of 8 A129 mice (4 group titration series) 24 hours prior to sub-cutaneous challenge with Zika virus PRVABC59. Plasma/sera samples were collected at regular intervals to track peripheral viremia and anti-zika antibody responses. FFPE tissues were collected at termination for histological analysis.
Immediately prior to challenge, human IgG was detectable in all infused animals. Within macaques the NT50 at this time was 250. All macaques that received plasma were protected against zika virus infection as determined by plasma/tissue qRT-PCR and IgM responses.
Titration within A129 mice gave a neutralisation titre of 110, above which mice were generally protected against zika infection. However this was not absolute as a small number of mice with high neutralisation levels were infected.
A protective NT50 of 250 has been identified for macaques and this further titrated in A129 mice to give a guide protective neutralisation titre of 110. The lack of protection in some mice with higher titres is currently unclear and studies are underway to compare their infection pathology with that of controls.
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Stage specific transcriptome profiling of castor bean tick Ixodes ricinus
Arthropod borne diseases are ubiquitously discussed topic whose relevance increases with ongoing changing climate, which extends the area of their incidence and affects profoundly the size of vector population, as well as its reproductive capacity, the abundance and spread of reservoir hosts and other variables that are generally tightly correlated with the spread of zoonoses.
The life cycle of arthropod borne pathogens are tightly bound to the life cycle of their host as well as to their vector organism. Therefore, the description of the vector life cycle should elucidate some questions related to the vector-pathogen dynamics including the factors important for successful disease transmission.
Tick has complex life cycle and for its completion it requires feeding on several host organisms, which is abused by the pathogen for its spreading within reservoir and host organisms.
Thus, more thorough description of factors driving tick developmental processes controlling its life cycle will be instrumental in understanding the nature of I. ricinus and its pathogens interactions and may also shed light on the process of blood feeding as an integral event in tick development as well as in potential pathogen transmission.
In our study we perform transcriptional profiling of all life stages of I. ricinus and provide a list of genes associated with particular life stages of I. ricinus and hence extend our knowledge in pursuit of potential acaricidal strategies.
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Stage specific transcriptome profiling of castor bean tick Ixodes ricinus
Arthropod borne diseases are ubiquitously discussed topic whose relevance increases with ongoing changing climate, which extends the area of their incidence and affects profoundly the size of vector population, as well as its reproductive capacity, the abundance and spread of reservoir hosts and other variables that are generally tightly correlated with the spread of zoonoses.
The life cycle of arthropod borne pathogens are tightly bound to the life cycle of their host as well as to their vector organism. Therefore, the description of the vector life cycle should elucidate some questions related to the vector-pathogen dynamics including the factors important for successful disease transmission.
Tick has complex life cycle and for its completion it requires feeding on several host organisms, which is abused by the pathogen for its spreading within reservoir and host organisms.
Thus, more thorough description of factors driving tick developmental processes controlling its life cycle will be instrumental in understanding the nature of I. ricinus and its pathogens interactions and may also shed light on the process of blood feeding as an integral event in tick development as well as in potential pathogen transmission.
In our study we perform transcriptional profiling of all life stages of I. ricinus and provide a list of genes associated with particular life stages of I. ricinus and hence extend our knowledge in pursuit of potential acaricidal strategies.
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Lumpy skin disease virus: transmission to dipteran vectors using animal and ex-vivo models
Lumpy skin disease (LSD) is a tropical neglected viral disease of cattle, characterised by numerous cutaneous lesions disseminated throughout the body. Historically endemic to the African continent, it has become a threat to Europe following the outbreaks of LSD in the Middle East and Eastern Europe.
LSD virus (LSDV) is a Capripoxvirus transmitted by insect vectors. Experimental and epidemiological studies have indicated a role for the stable fly (Stomoxys calcintrans) and the mosquito Aedes aegypti. Nevertheless the relative importance of these vector species and others is unclear. A study was designed to explore the risk of transmission of LSDV from cattle to different vector species including Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus. Cattle was challenged with LSDV to produce a bovine experimental model used as a natural source of LSDV to the potential vectors. Cattle samples were taken to quantify LSDV in different tissues and characterise the disease. All insect species were allowed to feed on LSDV-challenged cattle at regular intervals and incubated for up to eight days. This data was then used to model the dynamics of LSDV infection and transmission. All four species were able to acquire and maintain LSDV for up to eight days post feeding, and the risk of transmission from bovine donor to insect was dependent on the severity of the disease. A model was then generated using ex-vivo skin lesions and infectious blood that will allow further studies of the role of these vectors.
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Lumpy skin disease virus: transmission to dipteran vectors using animal and ex-vivo models
Lumpy skin disease (LSD) is a tropical neglected viral disease of cattle, characterised by numerous cutaneous lesions disseminated throughout the body. Historically endemic to the African continent, it has become a threat to Europe following the outbreaks of LSD in the Middle East and Eastern Europe.
LSD virus (LSDV) is a Capripoxvirus transmitted by insect vectors. Experimental and epidemiological studies have indicated a role for the stable fly (Stomoxys calcintrans) and the mosquito Aedes aegypti. Nevertheless the relative importance of these vector species and others is unclear. A study was designed to explore the risk of transmission of LSDV from cattle to different vector species including Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus. Cattle was challenged with LSDV to produce a bovine experimental model used as a natural source of LSDV to the potential vectors. Cattle samples were taken to quantify LSDV in different tissues and characterise the disease. All insect species were allowed to feed on LSDV-challenged cattle at regular intervals and incubated for up to eight days. This data was then used to model the dynamics of LSDV infection and transmission. All four species were able to acquire and maintain LSDV for up to eight days post feeding, and the risk of transmission from bovine donor to insect was dependent on the severity of the disease. A model was then generated using ex-vivo skin lesions and infectious blood that will allow further studies of the role of these vectors.
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Zika virus utilises the ubiquitin-proteasome pathway during infection of mosquito and human cells
Arthropod-borne viruses are able to infect vertebrate and invertebrate hosts. One such arbovirus is Zika virus (family Flaviviridae) that is mainly transmitted to humans by Aedes mosquitoes causing febrile illness and congenital Zika syndrome in infants. An interplay between host and virus proteins enables ZIKV to manipulate its host's cellular machineries in order to facilitate infection and evade antiviral responses. A possible mechanism it utilises is the ubiquitin-proteasome pathway (UPP) where target proteins are ubiquitinated and subsequently degraded by the proteasome. Results of proteomics analysis of Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C), anchored capsid (AC) or non-structural 3 (NS3) proteins revealed that these viral proteins interact with effector proteins of the UPP. One of these proteins is TER94 an AAA-ATPase that acts as a chaperone segregating ubiquitinated proteins to the proteasome complex. Knockdown experiments of TER94 or its human ortholog VCP using dsRNA or siRNAs showed reduced virus replication in AF5 or A549 cells. Using small molecule inhibitors of UPP proteins also diminished ZIKV replication. Inhibiting different stages of the pathway have identified critical steps during early stages of infection. The ubiquitination of lysine-rich ZIKV C and its interaction with TER94/VCP could be one of the many universal strategies that the virus employs when it switches between mosquito and human hosts. Understanding how ZIKV is able to infect both human and insect species could provide novel strategies for prevention and therapeutics.
Keywords: Zika virus, Aedes aegypti, ubiquitin-proteasome pathway, mosquito, virus-host interaction
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Zika virus utilises the ubiquitin-proteasome pathway during infection of mosquito and human cells
Arthropod-borne viruses are able to infect vertebrate and invertebrate hosts. One such arbovirus is Zika virus (family Flaviviridae) that is mainly transmitted to humans by Aedes mosquitoes causing febrile illness and congenital Zika syndrome in infants. An interplay between host and virus proteins enables ZIKV to manipulate its host's cellular machineries in order to facilitate infection and evade antiviral responses. A possible mechanism it utilises is the ubiquitin-proteasome pathway (UPP) where target proteins are ubiquitinated and subsequently degraded by the proteasome. Results of proteomics analysis of Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C), anchored capsid (AC) or non-structural 3 (NS3) proteins revealed that these viral proteins interact with effector proteins of the UPP. One of these proteins is TER94 an AAA-ATPase that acts as a chaperone segregating ubiquitinated proteins to the proteasome complex. Knockdown experiments of TER94 or its human ortholog VCP using dsRNA or siRNAs showed reduced virus replication in AF5 or A549 cells. Using small molecule inhibitors of UPP proteins also diminished ZIKV replication. Inhibiting different stages of the pathway have identified critical steps during early stages of infection. The ubiquitination of lysine-rich ZIKV C and its interaction with TER94/VCP could be one of the many universal strategies that the virus employs when it switches between mosquito and human hosts. Understanding how ZIKV is able to infect both human and insect species could provide novel strategies for prevention and therapeutics.
Keywords: Zika virus, Aedes aegypti, ubiquitin-proteasome pathway, mosquito, virus-host interaction
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St Abbs Head phlebovirus – a separate virus species or a strain of Uukuniemi phlebovirus?
St Abbs Head virus (SAHV), member of Phlebovirus genus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments.
SAHV was isolated from a pool of Ixodes uriae ticks collected at a seabird colony in Berwickshire, Scotland in 1979. There were quite a few related bunyaviruses found in tick and bird samples on the East Coast of Scotland and England in the 70s and 80s. Recently we have sequenced a sample of SAHV using next generation sequencing technology. The results suggested that this virus is very closely related to the Uukuniemi phlebovirus (UUKV). To determine how similar are SAHV and UUKV, we compared virus growth in various mammalian, bird and tick cell lines.
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St Abbs Head phlebovirus – a separate virus species or a strain of Uukuniemi phlebovirus?
St Abbs Head virus (SAHV), member of Phlebovirus genus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments.
SAHV was isolated from a pool of Ixodes uriae ticks collected at a seabird colony in Berwickshire, Scotland in 1979. There were quite a few related bunyaviruses found in tick and bird samples on the East Coast of Scotland and England in the 70s and 80s. Recently we have sequenced a sample of SAHV using next generation sequencing technology. The results suggested that this virus is very closely related to the Uukuniemi phlebovirus (UUKV). To determine how similar are SAHV and UUKV, we compared virus growth in various mammalian, bird and tick cell lines.
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Alphavirus E1 fusion protein: alternative conformations of the post-fusion trimer depending on the alphavirus
Background. Some of the best characterized alphaviruses are chikungunya virus (CHIKV), Semliki Forest virus (SFV) and Sindbis virus (SINV). E1 is a class II fusion protein, containing 3 domains (DI, DII, DIII) folded essentially as β-sheet, plus a stem region connecting DIII to the transmembrane (TM) segment. The fusion loop (FL) is at the tip of the elongated DII. The X-ray structure of the SFV E1 post-fusion trimer, truncated of the stem region, displayed a tripod-shape with the DII legs open and the FLs away from each other, contrary to the class II viral fusion proteins from other viral genera, in which the FLs interact at the tip of the post-fusion trimer. Here, we set to identify if the stem plays a structural role in zippering together the E1 trimer to bring the fusion loops into contact.
Methods. We produced the recombinant ectodomains of E1 of CHIKV, SINV and SFV containing or not the stem, crystallized them and determined the X-ray structure.
Results. We observed that CHIKV and SINV display E1 in closed conformation, in contrast to SFV, which displays a tripod even with the full stem. We identified a sequence motif in the stem responsible for the conformational difference.
Conclusion. Our results point to potential mechanistic differences between alphavirus E1 in driving fusion. Further functional studies are ongoing to pin-point the significance of these new findings, and the reasons for the alternative post-fusion conformations.
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