- 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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- Oral Abstract
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Chikungunya virus recruits the ESCRT trafficking machinery at multiple steps during the intracellular life cycle
Chikungunya virus (CHIKV) causes a re-emerging zoonotic disease characterized by fever and joint pain. Since there are no available vaccines nor medicine for CHIKV infection, better understanding of molecular mechanisms underlying CHIKV infection is demanded to develop new treatments. To date, few studies exist describing host factors necessary for intracellular trafficking of CHIKV; thus, the aim of this study is to identify trafficking genes regulating CHIKV infection and to investigate their roles.
We conducted imaging-based siRNA screen targeting 73 trafficking genes, examined the interaction with CHIKV proteins by co-immunoprecipitation and confocal analyses, and then investigated which infection steps of CHIKV were affected by the identified gene(s).
The siRNA screen showed that endosomal sorting complexes required for transport (ESCRT) proteins were involved in CHIKV infection. Co-immunoprecipitation analyses between ESCRT proteins and CHIKV proteins revealed that both structural and non-structural proteins of CHIKV interacted with HGS, a component of ESCRT-0 complex. Confocal analyses demonstrated colocalization of HGS with CHIKV E2 and dsRNA, a marker for the replicated CHIKV genome. Gene knockdown analyses using CHIKV replicon and CHIKV-like particle system demonstrated that HGS facilitated both genome replication and post-translational steps of CHIKV infection, as well as other ESCRT factors.
Here, we propose for the first time that CHIKV requires ESCRT factors at multiple steps during its intracellular life cycle.
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Chikungunya virus recruits the ESCRT trafficking machinery at multiple steps during the intracellular life cycle
Chikungunya virus (CHIKV) causes a re-emerging zoonotic disease characterized by fever and joint pain. Since there are no available vaccines nor medicine for CHIKV infection, better understanding of molecular mechanisms underlying CHIKV infection is demanded to develop new treatments. To date, few studies exist describing host factors necessary for intracellular trafficking of CHIKV; thus, the aim of this study is to identify trafficking genes regulating CHIKV infection and to investigate their roles.
We conducted imaging-based siRNA screen targeting 73 trafficking genes, examined the interaction with CHIKV proteins by co-immunoprecipitation and confocal analyses, and then investigated which infection steps of CHIKV were affected by the identified gene(s).
The siRNA screen showed that endosomal sorting complexes required for transport (ESCRT) proteins were involved in CHIKV infection. Co-immunoprecipitation analyses between ESCRT proteins and CHIKV proteins revealed that both structural and non-structural proteins of CHIKV interacted with HGS, a component of ESCRT-0 complex. Confocal analyses demonstrated colocalization of HGS with CHIKV E2 and dsRNA, a marker for the replicated CHIKV genome. Gene knockdown analyses using CHIKV replicon and CHIKV-like particle system demonstrated that HGS facilitated both genome replication and post-translational steps of CHIKV infection, as well as other ESCRT factors.
Here, we propose for the first time that CHIKV requires ESCRT factors at multiple steps during its intracellular life cycle.
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Novel flaviviruses of the ocean: Insights into the evolution and circulation of flaviviruses between marine invertebrate and vertebrate Hosts
Most described flaviviruses (family Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses has presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. In addition, we identified variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNA interference response. This suggests that Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono- and dinucleotide composition of marine flaviviruses compared to that of flaviviruses with known host status suggest that some marine flaviviruses share a nucleotide bias similar to that of vector-borne flaviviruses. Furthermore, we identify crustacean flavivirus endogenous viral elements that are closely related to elements of terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.
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Novel flaviviruses of the ocean: Insights into the evolution and circulation of flaviviruses between marine invertebrate and vertebrate Hosts
Most described flaviviruses (family Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses has presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. In addition, we identified variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNA interference response. This suggests that Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono- and dinucleotide composition of marine flaviviruses compared to that of flaviviruses with known host status suggest that some marine flaviviruses share a nucleotide bias similar to that of vector-borne flaviviruses. Furthermore, we identify crustacean flavivirus endogenous viral elements that are closely related to elements of terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.
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Modulation of arbovirus infection by mosquito saliva
Arboviruses constitute a major public health problem; in particular mosquito-borne arboviruses that continuously emerge and re-emerge. Arbovirus infection of mammals is enhanced by the presence of a mosquito-bite at the inoculation site, or by the co-inoculation of extracted mosquito saliva alongside virus, in comparison to virus experimentally administered by needle inoculation in the absence of bite/saliva. Inflammatory responses elicited to saliva appear to be key in facilitating this enhancement. As such, we have studied the mechanistic basis for these observations by investigating mosquito-bite factors, as well as host responses, involved in facilitating viral enhancement. We have studied whether saliva from different mosquito species successfully enhance virus infection. Interestingly, while saliva from Aedes genus enhanced virus infection, An. gambiae saliva did not. This could partly explain why An. gambiae mosquitos are unsuitable vectors for transmitting most arboviruses. By comparing the effects that saliva from these different species have at the inoculation-site, we have further specified which inflammatory responses modulate arbovirus infection in the skin. Using an in vivo mouse-model we demonstrate that An. gambiae causes significantly less oedema than Ae. aegypti and that histamine induced oedema in the absence of salivary-factors also enhances infection. Also, measuring cytokine responses to Aedes and Anopheles saliva, showed that several key anti-viral chemokines such as CCL5 were significantly more upregulated by Anopheles. Hence, we’re providing important insights into how mosquito saliva modulates infection. A better understanding of this will aid the development of anti-viral treatments targeting factors within the mosquito bite that are common to many distinct infections.
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Modulation of arbovirus infection by mosquito saliva
Arboviruses constitute a major public health problem; in particular mosquito-borne arboviruses that continuously emerge and re-emerge. Arbovirus infection of mammals is enhanced by the presence of a mosquito-bite at the inoculation site, or by the co-inoculation of extracted mosquito saliva alongside virus, in comparison to virus experimentally administered by needle inoculation in the absence of bite/saliva. Inflammatory responses elicited to saliva appear to be key in facilitating this enhancement. As such, we have studied the mechanistic basis for these observations by investigating mosquito-bite factors, as well as host responses, involved in facilitating viral enhancement. We have studied whether saliva from different mosquito species successfully enhance virus infection. Interestingly, while saliva from Aedes genus enhanced virus infection, An. gambiae saliva did not. This could partly explain why An. gambiae mosquitos are unsuitable vectors for transmitting most arboviruses. By comparing the effects that saliva from these different species have at the inoculation-site, we have further specified which inflammatory responses modulate arbovirus infection in the skin. Using an in vivo mouse-model we demonstrate that An. gambiae causes significantly less oedema than Ae. aegypti and that histamine induced oedema in the absence of salivary-factors also enhances infection. Also, measuring cytokine responses to Aedes and Anopheles saliva, showed that several key anti-viral chemokines such as CCL5 were significantly more upregulated by Anopheles. Hence, we’re providing important insights into how mosquito saliva modulates infection. A better understanding of this will aid the development of anti-viral treatments targeting factors within the mosquito bite that are common to many distinct infections.
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Virus-host interactions for Bluetongue virus and characterization of a new NS3 function
Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of ruminants. Infections by this virus result in variable clinical outcomes depending on host and viral factors. We know that these variances largely rely on virus-host molecular interactions. To investigate these networks, our lab has developed since few years an interactomic research programme based on the yeast two-hybrid approach.To date, we have carried out the complete BTV interactome against two complementary DNA libraries, from bovine and culicoides cell lines, that allowed to the identification of about one hundred new putative cellular partners for BTV. We are now validating these interactions through biochemical and functional studies. To complete this interactomic programme, we now also combine affinity purification approach coupled to mass spectrometry. Upon immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified new cellular interactors including the serine/threonine kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate this pathway is conserved throughout multiple serotypes but appears to be specific to BTV compared to other orbiviruses. Inhibition of MAPK/ERK pathway activation with the pharmacological molecule U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.
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Virus-host interactions for Bluetongue virus and characterization of a new NS3 function
Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of ruminants. Infections by this virus result in variable clinical outcomes depending on host and viral factors. We know that these variances largely rely on virus-host molecular interactions. To investigate these networks, our lab has developed since few years an interactomic research programme based on the yeast two-hybrid approach.To date, we have carried out the complete BTV interactome against two complementary DNA libraries, from bovine and culicoides cell lines, that allowed to the identification of about one hundred new putative cellular partners for BTV. We are now validating these interactions through biochemical and functional studies. To complete this interactomic programme, we now also combine affinity purification approach coupled to mass spectrometry. Upon immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified new cellular interactors including the serine/threonine kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate this pathway is conserved throughout multiple serotypes but appears to be specific to BTV compared to other orbiviruses. Inhibition of MAPK/ERK pathway activation with the pharmacological molecule U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.
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Budding induces symmetry defects in flavivirus virions
Flaviviruses assemble at the endoplasmic reticulum in an immature, non-infectious state that is designed to inhibit premature fusion with the host. Maturation of the particle occurs when it enters the trans-Golgi network, where low pH induces a conformational rearrangement of the virus glycoproteins. This transition causes 60 prM-E glycoprotein trimers in the immature virus to rearrange into 90 M-E dimers in the mature virus. We hypothesize that flaviviruses do not have exact icosahedral symmetry, thus providing flexibility for the large conformational rearrangements that are required during the virus life cycle. When icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of immature Kunjin virus (KUNV), a low virulent strain of West Nile virus, the nucleocapsid core touched the inside of the viral lipid membrane at the “proximal pole” and was asymmetrically positioned within the lipid bilayer envelope. The outer glycoprotein spikes on the “distal pole” were either distorted or missing. In the asymmetric reconstruction of mature KUNV, the core was re-positioned, as expected, concentric with the glycoprotein shell and there remained a distortion of the glycoproteins on one pole of the virion. This suggests that the interactions between the core and glycoproteins are altered during viral assembly and budding from the ER. In addition, it implies that the glycoproteins have a geometric defect that perhaps facilitates the transitions that occur during maturation. This defect in number and arrangement of the glycoproteins may reflect the consequence of membrane budding.
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Budding induces symmetry defects in flavivirus virions
Flaviviruses assemble at the endoplasmic reticulum in an immature, non-infectious state that is designed to inhibit premature fusion with the host. Maturation of the particle occurs when it enters the trans-Golgi network, where low pH induces a conformational rearrangement of the virus glycoproteins. This transition causes 60 prM-E glycoprotein trimers in the immature virus to rearrange into 90 M-E dimers in the mature virus. We hypothesize that flaviviruses do not have exact icosahedral symmetry, thus providing flexibility for the large conformational rearrangements that are required during the virus life cycle. When icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of immature Kunjin virus (KUNV), a low virulent strain of West Nile virus, the nucleocapsid core touched the inside of the viral lipid membrane at the “proximal pole” and was asymmetrically positioned within the lipid bilayer envelope. The outer glycoprotein spikes on the “distal pole” were either distorted or missing. In the asymmetric reconstruction of mature KUNV, the core was re-positioned, as expected, concentric with the glycoprotein shell and there remained a distortion of the glycoproteins on one pole of the virion. This suggests that the interactions between the core and glycoproteins are altered during viral assembly and budding from the ER. In addition, it implies that the glycoproteins have a geometric defect that perhaps facilitates the transitions that occur during maturation. This defect in number and arrangement of the glycoproteins may reflect the consequence of membrane budding.
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Orthobunyavirus recruits host ESCRT machinery to Golgi compartments to achieve efficient viral particle production
Oropouche virus (OROV) is the etiological agent of Oropouche fever, a febrile illness frequent in South America. Despite its importance, the replicative cycle of this Orthobunyavirus is only starting to emerge. In this study we described the assembly pathway of OROV in Hela cells. After a thorough description of OROV one-step replication cycle, we observed by Immunofluorescence large vesicle-like structures enriched in OROV proteins termed viral factories (Vfs). These vesicles were associated with cis-Golgi (Giantin), trans-Golgi network (TGN46) and endosomal (HRS) proteins. Immuno-EM analysis of these Vfs revealed a resemblance with multivesicular body structure, which also contained viral particles. Interestingly, no leakage of late endosomal proteins (CD63 and Lamp1) was observed associated with Vfs. Therefore, we looked if ESCRT (Endosomal Sorting Complexes required for Transport) machinery could play a role in OROV replication. Depletion of Tsg101 (ESCRT-I) and Alix by siRNA compromised Vfs formation (∼40% in both cases) that contained a significantly lower number of intra-luminal viral-like particles (∼50% in both cases). Additionally, depletion of Alix led to a strong reduction in viral production (∼80%). Moreover, the overexpression of a dominant negative form of the AAATPase Vps4A (Vps4E/Q), which disrupts the MVB pathway, led to an enlargement in Vfs’ area (∼119%), where Vps4E/Q accumulated. Importantly, we also detected a colocalization between Vps4E/Q/TGN46 and Alix/TGN46 in viral factories, suggesting an involvement of ESCRT machinery, which is re-localized to the TGN in virus assembly. Therefore, our data represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.
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Orthobunyavirus recruits host ESCRT machinery to Golgi compartments to achieve efficient viral particle production
Oropouche virus (OROV) is the etiological agent of Oropouche fever, a febrile illness frequent in South America. Despite its importance, the replicative cycle of this Orthobunyavirus is only starting to emerge. In this study we described the assembly pathway of OROV in Hela cells. After a thorough description of OROV one-step replication cycle, we observed by Immunofluorescence large vesicle-like structures enriched in OROV proteins termed viral factories (Vfs). These vesicles were associated with cis-Golgi (Giantin), trans-Golgi network (TGN46) and endosomal (HRS) proteins. Immuno-EM analysis of these Vfs revealed a resemblance with multivesicular body structure, which also contained viral particles. Interestingly, no leakage of late endosomal proteins (CD63 and Lamp1) was observed associated with Vfs. Therefore, we looked if ESCRT (Endosomal Sorting Complexes required for Transport) machinery could play a role in OROV replication. Depletion of Tsg101 (ESCRT-I) and Alix by siRNA compromised Vfs formation (∼40% in both cases) that contained a significantly lower number of intra-luminal viral-like particles (∼50% in both cases). Additionally, depletion of Alix led to a strong reduction in viral production (∼80%). Moreover, the overexpression of a dominant negative form of the AAATPase Vps4A (Vps4E/Q), which disrupts the MVB pathway, led to an enlargement in Vfs’ area (∼119%), where Vps4E/Q accumulated. Importantly, we also detected a colocalization between Vps4E/Q/TGN46 and Alix/TGN46 in viral factories, suggesting an involvement of ESCRT machinery, which is re-localized to the TGN in virus assembly. Therefore, our data represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.
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Developing a pseudotype model to test infectivity among different Zika virus mutants
Viral infections pose a massive threat to the public health; recently several viral outbreaks had occurred across the globe, including Ebola virus, Middle East respiratory syndrome and Zika virus (ZIKV). ZIKV had great importance due to the appearance of malformation and neurological syndromes in newborns. One of the things that are worth noting is that ZIKV has been present in nature since the second half of the 1900s but only until the outbreak of 2015 in Brazil it gained a prominent spot in the public health surveillance.
The main goal of the project was to develop a suitable method to test different mutations that occurred naturally throughout several outbreaks of the virus across the globe, with particular emphasis in the Asian and American epidemics between 2007 and 2016. To achieve the objectives, the strategy previously used in other viruses known as pseudotyping was proposed. Pseudotyping poses a safe alternative to life virus work due to the lack of a productive infectious cycle, the use of a retroviral backbone supplemented with the glycoprotein of interest has been used in hazardous viruses such as Ebola or Hepatitis C. This system also allows to test individual mutations and the impact in the infectivity of the virus discarding any other accessory protein within the wild type virus. HIV-1 and MLV backbones were tested using a matrix of different ratios of glycoprotein and retrovirus plasmid to find the best set of concentrations; different cell lines were also tested, and protein expression viral proteins were detected.
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Developing a pseudotype model to test infectivity among different Zika virus mutants
Viral infections pose a massive threat to the public health; recently several viral outbreaks had occurred across the globe, including Ebola virus, Middle East respiratory syndrome and Zika virus (ZIKV). ZIKV had great importance due to the appearance of malformation and neurological syndromes in newborns. One of the things that are worth noting is that ZIKV has been present in nature since the second half of the 1900s but only until the outbreak of 2015 in Brazil it gained a prominent spot in the public health surveillance.
The main goal of the project was to develop a suitable method to test different mutations that occurred naturally throughout several outbreaks of the virus across the globe, with particular emphasis in the Asian and American epidemics between 2007 and 2016. To achieve the objectives, the strategy previously used in other viruses known as pseudotyping was proposed. Pseudotyping poses a safe alternative to life virus work due to the lack of a productive infectious cycle, the use of a retroviral backbone supplemented with the glycoprotein of interest has been used in hazardous viruses such as Ebola or Hepatitis C. This system also allows to test individual mutations and the impact in the infectivity of the virus discarding any other accessory protein within the wild type virus. HIV-1 and MLV backbones were tested using a matrix of different ratios of glycoprotein and retrovirus plasmid to find the best set of concentrations; different cell lines were also tested, and protein expression viral proteins were detected.
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Host genome depletion to determine the evolution, genetic diversity and transmission patterns of full genome sequences of African swine fever genotype IX from Uganda
African swine fever (ASF) is a highly contagious viral haemorrhagic disease of domestic pigs, wild boar and feral swine. It is caused by the only DNA arbovirus, African swine fever virus (ASFV), which is the only member of the family Asfarviridae and is transmitted by the soft ticks in the genus Ornithodoros. ASF results in mortalities of up to 100% and exhibits different epidemiological patterns in Africa, Europe and Asia. This ASFV has recently spread to other countries in Europe and is also now present in Asia where it has spread like wildfire, resulting into high economic losses. The lack of a treatment or vaccine against this disease makes its control and prevention extremely difficult. This is partly due to the large size of the ASFV genome (170-190 kbp) and the many genes (187–190) that it encodes. There are currently 24 genotypes, with genotype IX predominating in Uganda and Kenya. Understanding the genetic characteristics of ASFV have focused mainly on partial fragments of selected genes, thus missing the opportunity to investigate the evolution, genetic diversity and patterns of transmission at the whole genomic level. Using the host-genome depletion approach we show an increase in the amount of ASFV DNA recovered directly from clinical material and generate genotype IX complete genomes on an NGS platform. We further show the inter-epidemic relationships of ASFV outbreaks in Uganda. The data contribute towards understanding the patterns and dynamics of transmission and maintenance of ASFV in a country in which ASF is endemic.
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Host genome depletion to determine the evolution, genetic diversity and transmission patterns of full genome sequences of African swine fever genotype IX from Uganda
African swine fever (ASF) is a highly contagious viral haemorrhagic disease of domestic pigs, wild boar and feral swine. It is caused by the only DNA arbovirus, African swine fever virus (ASFV), which is the only member of the family Asfarviridae and is transmitted by the soft ticks in the genus Ornithodoros. ASF results in mortalities of up to 100% and exhibits different epidemiological patterns in Africa, Europe and Asia. This ASFV has recently spread to other countries in Europe and is also now present in Asia where it has spread like wildfire, resulting into high economic losses. The lack of a treatment or vaccine against this disease makes its control and prevention extremely difficult. This is partly due to the large size of the ASFV genome (170-190 kbp) and the many genes (187–190) that it encodes. There are currently 24 genotypes, with genotype IX predominating in Uganda and Kenya. Understanding the genetic characteristics of ASFV have focused mainly on partial fragments of selected genes, thus missing the opportunity to investigate the evolution, genetic diversity and patterns of transmission at the whole genomic level. Using the host-genome depletion approach we show an increase in the amount of ASFV DNA recovered directly from clinical material and generate genotype IX complete genomes on an NGS platform. We further show the inter-epidemic relationships of ASFV outbreaks in Uganda. The data contribute towards understanding the patterns and dynamics of transmission and maintenance of ASFV in a country in which ASF is endemic.
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- Poster Abstract
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Mapping the virome in lab-reared and wild-caught Aedes albopictus mosquitoes
Mosquitoes are known vectors that can harbor a wide array of viruses that range from classical arboviruses that can infect vertebrates to insect-specific viruses that are restricted only in mosquito. Recent advances in the field of metagenomics and bioinformatics have strongly supported studies on mosquito-associated viruses. However, the study for mosquito virome profile is still inadequate. In the present study, we aimed to investigate and compare the virome structure and profile between laboratory reared and wild-caught Aedes albopictus mosquito. Four mosquito samples (egg, larvae, pupae, adult) of laboratory rearedand two (larvae and adult)of field collected from Guangzhou of China were prepared for next generation sequencing (NGS) and analyzed. The results indicated that the common viruses presented in all samples were High Island virus, Aedes flavivirus, and Guato virus, whereas the Wenzhou sobemo-like virus 4 and Hubei mosquito virus 2 were the most dominant virus and presented only in wild mosquito samples. Additionally, unclassified Papillomaviridae viruses was the most dominant vertebrate virus, which was significantly detected only in wild adult mosquitoes. Important to note, the viral composition of larvae obtained from the wild was the most diverse group with many viruses belonging to virus families including Flaviviridae, Totiviridae, Tymoviridae, Rhabdoviridae, Orthomyxoviridae, Iflaviridae and unclassified viruses. This observed diverse nature can be attributed to influence of environment substrates and water in which the mosquito larvae inhabit. In conclusion, our findings provide essential information necessary in understanding the structure and diversity of Aedes virome.
Keywords: mosquito, virome, Aedes albopictus, next generation sequencing.
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Mapping the virome in lab-reared and wild-caught Aedes albopictus mosquitoes
Mosquitoes are known vectors that can harbor a wide array of viruses that range from classical arboviruses that can infect vertebrates to insect-specific viruses that are restricted only in mosquito. Recent advances in the field of metagenomics and bioinformatics have strongly supported studies on mosquito-associated viruses. However, the study for mosquito virome profile is still inadequate. In the present study, we aimed to investigate and compare the virome structure and profile between laboratory reared and wild-caught Aedes albopictus mosquito. Four mosquito samples (egg, larvae, pupae, adult) of laboratory rearedand two (larvae and adult)of field collected from Guangzhou of China were prepared for next generation sequencing (NGS) and analyzed. The results indicated that the common viruses presented in all samples were High Island virus, Aedes flavivirus, and Guato virus, whereas the Wenzhou sobemo-like virus 4 and Hubei mosquito virus 2 were the most dominant virus and presented only in wild mosquito samples. Additionally, unclassified Papillomaviridae viruses was the most dominant vertebrate virus, which was significantly detected only in wild adult mosquitoes. Important to note, the viral composition of larvae obtained from the wild was the most diverse group with many viruses belonging to virus families including Flaviviridae, Totiviridae, Tymoviridae, Rhabdoviridae, Orthomyxoviridae, Iflaviridae and unclassified viruses. This observed diverse nature can be attributed to influence of environment substrates and water in which the mosquito larvae inhabit. In conclusion, our findings provide essential information necessary in understanding the structure and diversity of Aedes virome.
Keywords: mosquito, virome, Aedes albopictus, next generation sequencing.
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A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission
Although dengue is the most prevalent arthropod-borne viral disease in humans, no effective medication or vaccine is presently available. Previous studies suggested that mosquito salivary proteins influence infection by the dengue virus (DENV) in the mammalian host. However, the effects of salivary proteins on DENV replication within the Aedes aegypti mosquito remain largely unknown. In this study, we investigated the effect of a specific salivary protein (named AaSG34) on DENV serotype 2 (DENV2) replication and transmission. We showed that transcripts of AaSG34 were upregulated in the salivary glands of Aedes aegypti mosquitoes after a meal of blood infected with DENV2. Transcripts of the dengue viral genome and envelop protein in the salivary glands were significantly diminished after an infectious blood meal when AaSG34 was silenced. The effect of AaSG34 on DENV2 transmission was investigated in Stat1-deficient mice. The intradermal inoculation of infectious mosquito saliva induced hemorrhaging inthe Stat1-deficient mice; however, saliva from the AaSG34-silenced mosquitoes did not induce hemorrhaging, suggesting that AaSG34 enhances DENV2 transmission. This is the first report to demonstrate that the protein AaSG34 promotes DENV2 replication in mosquito salivary glands and enhances the transmission of the virus to the mammalian host.
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A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission
Although dengue is the most prevalent arthropod-borne viral disease in humans, no effective medication or vaccine is presently available. Previous studies suggested that mosquito salivary proteins influence infection by the dengue virus (DENV) in the mammalian host. However, the effects of salivary proteins on DENV replication within the Aedes aegypti mosquito remain largely unknown. In this study, we investigated the effect of a specific salivary protein (named AaSG34) on DENV serotype 2 (DENV2) replication and transmission. We showed that transcripts of AaSG34 were upregulated in the salivary glands of Aedes aegypti mosquitoes after a meal of blood infected with DENV2. Transcripts of the dengue viral genome and envelop protein in the salivary glands were significantly diminished after an infectious blood meal when AaSG34 was silenced. The effect of AaSG34 on DENV2 transmission was investigated in Stat1-deficient mice. The intradermal inoculation of infectious mosquito saliva induced hemorrhaging inthe Stat1-deficient mice; however, saliva from the AaSG34-silenced mosquitoes did not induce hemorrhaging, suggesting that AaSG34 enhances DENV2 transmission. This is the first report to demonstrate that the protein AaSG34 promotes DENV2 replication in mosquito salivary glands and enhances the transmission of the virus to the mammalian host.
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A novel class of small molecule inhibitors targeting the chikungunya virus capping machinery with a high barrier to resistance
Background: Despite the worldwide re-emergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we identified a novel class of CHIKV inhibitors i.e. the CHVB series.
Methods: CPE-reduction and virus yield assays were performed in Vero cells. Drug-resistant variants were selected using clonal resistance selection. The enzymatic assays for alphavirus capping were done using the non-structural protein 1 (nsP1) of Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV).
Results: CHVB compounds inhibited the in vitro replication of CHIKV isolates with EC50 values in the low μM range. In virus yield assays, the most potent analogues reduced the viral load with 4-5 log10. CHVB-resistant variants were selected and found to carry (i) two mutations in the gene encoding nsP1 (responsible for viral RNA capping), (ii) one mutation in nsP2 and (iii) one mutation in nsP3. Reverse-engineering suggested that nsP1 is the target of CHVB, since both nsP1 mutations were needed to achieve 10-fold resistance. Interestingly, the CHVBres virus proved cross-resistant to the MADTP series, a class of CHIKV capping inhibitors that we described earlier, suggesting a similar mode of action. In enzymatic assays, CHVB proved a potent inhibitor of the methyltransferase and guanylyltransferase activities of nsP1 of SFV and VEEV.
Conclusion: We identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity and the high barrier to resistance make this chemical scaffold a potential candidate for CHIKV drug development.
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A novel class of small molecule inhibitors targeting the chikungunya virus capping machinery with a high barrier to resistance
Background: Despite the worldwide re-emergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we identified a novel class of CHIKV inhibitors i.e. the CHVB series.
Methods: CPE-reduction and virus yield assays were performed in Vero cells. Drug-resistant variants were selected using clonal resistance selection. The enzymatic assays for alphavirus capping were done using the non-structural protein 1 (nsP1) of Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV).
Results: CHVB compounds inhibited the in vitro replication of CHIKV isolates with EC50 values in the low μM range. In virus yield assays, the most potent analogues reduced the viral load with 4-5 log10. CHVB-resistant variants were selected and found to carry (i) two mutations in the gene encoding nsP1 (responsible for viral RNA capping), (ii) one mutation in nsP2 and (iii) one mutation in nsP3. Reverse-engineering suggested that nsP1 is the target of CHVB, since both nsP1 mutations were needed to achieve 10-fold resistance. Interestingly, the CHVBres virus proved cross-resistant to the MADTP series, a class of CHIKV capping inhibitors that we described earlier, suggesting a similar mode of action. In enzymatic assays, CHVB proved a potent inhibitor of the methyltransferase and guanylyltransferase activities of nsP1 of SFV and VEEV.
Conclusion: We identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity and the high barrier to resistance make this chemical scaffold a potential candidate for CHIKV drug development.
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The piRNA pathway in host-pathogen interaction: Aedes albopictus and arboviruses
Aedes albopictus is an aggressive invasive species and a competent vector for over 20 arboviruses, including Chikungunya, Dengue and Zika viruses. Understanding the molecular and cellular interactions between viruses and vectors is key to implement transmission-blocking strategies to prevent viral outbreaks. However, the mechanisms that shape vector competence are poorly understood. Recent evidence reveals that the genomes of Aedes spp. harbour fragmented viral sequences which produce PIWI-interacting RNAs (piRNAs), suggesting a role in vector competence. Current knowledge of the piRNA pathway in Ae. albopictus is limited, and its possible role in the establishment of persistent infections widely unknown.
We combined cutting-edge bioinformatic analyses based on next-generation sequencing data with molecular biology and virology techniques to characterise the main genes of the piRNA pathway in this mosquito species, assess their polymorphisms and analyse their expression throughout mosquito development and following infection with the Chikungunya and Dengue-1 viruses.
We identified seven piwi genes which displayed high levels of polymorphism across populations and signs of adaptive evolution. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with sites under positive selection. Infection experiments indicated specific responses depending on viral species, time of infection and mosquito tissue, highlighting distinct roles for specific Piwi proteins.
In conclusion, this work helps define the role of the piRNA pathway in persistent arboviral infections and understand the evolutionary divergence among piwi proteins.
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The piRNA pathway in host-pathogen interaction: Aedes albopictus and arboviruses
Aedes albopictus is an aggressive invasive species and a competent vector for over 20 arboviruses, including Chikungunya, Dengue and Zika viruses. Understanding the molecular and cellular interactions between viruses and vectors is key to implement transmission-blocking strategies to prevent viral outbreaks. However, the mechanisms that shape vector competence are poorly understood. Recent evidence reveals that the genomes of Aedes spp. harbour fragmented viral sequences which produce PIWI-interacting RNAs (piRNAs), suggesting a role in vector competence. Current knowledge of the piRNA pathway in Ae. albopictus is limited, and its possible role in the establishment of persistent infections widely unknown.
We combined cutting-edge bioinformatic analyses based on next-generation sequencing data with molecular biology and virology techniques to characterise the main genes of the piRNA pathway in this mosquito species, assess their polymorphisms and analyse their expression throughout mosquito development and following infection with the Chikungunya and Dengue-1 viruses.
We identified seven piwi genes which displayed high levels of polymorphism across populations and signs of adaptive evolution. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with sites under positive selection. Infection experiments indicated specific responses depending on viral species, time of infection and mosquito tissue, highlighting distinct roles for specific Piwi proteins.
In conclusion, this work helps define the role of the piRNA pathway in persistent arboviral infections and understand the evolutionary divergence among piwi proteins.
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Discovery of novel endogenous viral elements in Aedes spp. mosquitoes
More LessThe genomes of Aedes spp. mosquitoes contain integrated sequences from nonretroviral RNA viruses that are enriched in piRNA clusters, are embedded next to transposable elements (TEs) and produce piRNAs. The parallelism between TEs and viral integrations led to the hypothesis that viral integrations may constitute an archive of past viral infections and potentially have an immunity impact on novel infection with cognate viruses, similarly to how the piRNA pathway interacts with TEs. A corollary of this hypothesis is that the landscape of viral integrations should be variable across populations depending on their viral exposure. The highly repetitive nature of Aedes spp. genomes make the discovery of viral integrations from whole genome sequencing data of wild mosquitoes a daunting task.
Here we describe a novel bioinformatic pipeline to rigorously identify viral integrations using Next Generation Sequencing (NGS) data. Libraries from single or pools of mosquitoes, reference genome statistics, the landscape of TEs and the geographic origin of the analyzed samples are the actors of the analysis.
This pipeline has been tested in Ae. aegypti and Ae. albopictus mosquitoes, allowing to compare the performance of the analyses on genome assemblies of different completeness. We identified novel viral integrations in both genomes. Additionally, we show that the landscape of viral integrations is dynamic, with a population-specific behavior that we can leverage to formulate hypothesis on mechanisms of integration and the biological role of viral integrations.
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Assessment of tick-borne flavivirus host factors through genome-scale screens
Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular biology of the POWV replication cycle in mammalian cells, we performed genome-scale screens to uncover host factors required for viral replication. Putative proviral host factors were identified by infecting pools of cells containing knockout mutations in non-essential genes with POWV, followed by analysis of cells resistant to virus-induced cell death. Many endoplasmic reticulum membrane complex proteins were revealed in these screens, suggesting that TBFVs share some common host cell hijacking strategies with mosquito-borne flaviviruses. Candidate proteins that function in cell-matrix adhesion, glycosylation, or RNA binding in uninfected cells were the focus of validation studies. We used single-gene knockout cell lines to investigate possible proviral roles for specific proteins in the replication cycle of either POWV or Langat virus (LGTV), a non-pathogenic and model TBFV. Proteins identified in the POWV screens were not necessarily critical for LGTV replication. These results suggest that our screens were able to identify both pan-flaviviral, as well as POWV-specific, host gene products exploited during virus replication. Ongoing work is focused on characterizing distinct host-cell requirements of diverse flaviviruses. This genetic assessment of POWV replication factors, in combination with ensuing mechanistic studies, will provide possible avenues for the development of host-targeting countermeasures.
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Assessment of tick-borne flavivirus host factors through genome-scale screens
Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular biology of the POWV replication cycle in mammalian cells, we performed genome-scale screens to uncover host factors required for viral replication. Putative proviral host factors were identified by infecting pools of cells containing knockout mutations in non-essential genes with POWV, followed by analysis of cells resistant to virus-induced cell death. Many endoplasmic reticulum membrane complex proteins were revealed in these screens, suggesting that TBFVs share some common host cell hijacking strategies with mosquito-borne flaviviruses. Candidate proteins that function in cell-matrix adhesion, glycosylation, or RNA binding in uninfected cells were the focus of validation studies. We used single-gene knockout cell lines to investigate possible proviral roles for specific proteins in the replication cycle of either POWV or Langat virus (LGTV), a non-pathogenic and model TBFV. Proteins identified in the POWV screens were not necessarily critical for LGTV replication. These results suggest that our screens were able to identify both pan-flaviviral, as well as POWV-specific, host gene products exploited during virus replication. Ongoing work is focused on characterizing distinct host-cell requirements of diverse flaviviruses. This genetic assessment of POWV replication factors, in combination with ensuing mechanistic studies, will provide possible avenues for the development of host-targeting countermeasures.
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Molecular determinants of dengue virus infection in Aedes aegypti midgut
To be successfully transmitted to another susceptible human host by Aedes aegypti, dengue virus (DENV) must first successfully infect the mosquito midgut. The virus-host interactions that enable successful midgut infection, however, is not well understood. To understand the important interactions for successful midgut infection, we took advantage of the wild-type DENV2 16681 and its attenuated derivative PDK53, which has been shown to be refractory in mosquito infection. Using oral infectious-blood feeding, we observed that PDK53 failed to produce infectious progenies in the midgut, despite detectable viral genome replication. Furthermore, we found that the foci of PDK53 infection in the midgut, detected by immunofluorescence staining, was limited in both size and number, compared to its parent, 16681. Transcriptional analysis of the mosquito midgut revealed increased expression of genes in multiple innate immune pathways upon PDK53 infection but not 16681. To pinpoint the mutation responsible for this phenotype, we constructed an infectious clone of 16681 and used site-directed mutagenesis to substitute each of the known mutations in PDK53 into the 16681 genomic backbone. This approach pinpointed the NS1 G53D mutation as the single most important attenuating mutation in PDK53 in engendering refractoriness to mosquito midgut infection. Mechanistically, our data also suggests that this mutation affected the virus-ER-resident protein interactions that impacted the efficiency of DENV replication and hence induction of the innate immune response. Our findings reveal insights into the pathogenesis of dengue and adds to the body of knowledge on critical virushost interactions that govern epidemiological fitness of DENV.
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Molecular determinants of dengue virus infection in Aedes aegypti midgut
To be successfully transmitted to another susceptible human host by Aedes aegypti, dengue virus (DENV) must first successfully infect the mosquito midgut. The virus-host interactions that enable successful midgut infection, however, is not well understood. To understand the important interactions for successful midgut infection, we took advantage of the wild-type DENV2 16681 and its attenuated derivative PDK53, which has been shown to be refractory in mosquito infection. Using oral infectious-blood feeding, we observed that PDK53 failed to produce infectious progenies in the midgut, despite detectable viral genome replication. Furthermore, we found that the foci of PDK53 infection in the midgut, detected by immunofluorescence staining, was limited in both size and number, compared to its parent, 16681. Transcriptional analysis of the mosquito midgut revealed increased expression of genes in multiple innate immune pathways upon PDK53 infection but not 16681. To pinpoint the mutation responsible for this phenotype, we constructed an infectious clone of 16681 and used site-directed mutagenesis to substitute each of the known mutations in PDK53 into the 16681 genomic backbone. This approach pinpointed the NS1 G53D mutation as the single most important attenuating mutation in PDK53 in engendering refractoriness to mosquito midgut infection. Mechanistically, our data also suggests that this mutation affected the virus-ER-resident protein interactions that impacted the efficiency of DENV replication and hence induction of the innate immune response. Our findings reveal insights into the pathogenesis of dengue and adds to the body of knowledge on critical virushost interactions that govern epidemiological fitness of DENV.
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Establishment of a stable subgenomic dengue virus type 1 replicon system in Aedes albopictus mosquito cells for identification of DENV transmission blocking molecules
To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets.
In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.
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Establishment of a stable subgenomic dengue virus type 1 replicon system in Aedes albopictus mosquito cells for identification of DENV transmission blocking molecules
To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets.
In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.
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National Collection of Pathogenic Viruses: A Repository for Well Characterised and Authenticated Viruses
The National Collection of Pathogenic Viruses (NCPV) is one of four repositories making up Public Health England Culture Collections. It was established two decades ago to offer safe and secure facilities for storage of patents and deposits as well as to supply standardised virus products to the wider scientific community. Within the collection are strains of both medical and veterinary importance, with representation from a wide variety of virus families, classified as Hazard Groups 2, 3 and 4 by the Advisory Committee for Dangerous Pathogens (ACDP). These include arboviruses such as Dengue, Yellow-fever, Oropouche and Sandfly fever Naples.
Throughout the year, the collection receives virus deposits. The depositing process is free and simple. The quality of every virus material received in the collection is assessed through a series of tests to confirm viability and absence of microbial contamination (including mycoplasmas). Virus identity is confirmed by nucleic acid profiling. Once standards are met, the virus products are made available to the wider scientific community. In addition to facilitating access to viruses and their associated products as well as secure storage for patents, NCPV also undertakes collaborative projects and contract research/developmental work with academic and commercial partners, by providing expertise in the handling of viral pathogens.
A repository for well-characterised, authenticated viruses is a useful resource for the scientific community, aiding in the understanding of circulating viral pathogens and providing access to quality-assured reference reagents for the identification and development of vaccines and therapeutics in the event of global viral outbreaks.
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National Collection of Pathogenic Viruses: A Repository for Well Characterised and Authenticated Viruses
The National Collection of Pathogenic Viruses (NCPV) is one of four repositories making up Public Health England Culture Collections. It was established two decades ago to offer safe and secure facilities for storage of patents and deposits as well as to supply standardised virus products to the wider scientific community. Within the collection are strains of both medical and veterinary importance, with representation from a wide variety of virus families, classified as Hazard Groups 2, 3 and 4 by the Advisory Committee for Dangerous Pathogens (ACDP). These include arboviruses such as Dengue, Yellow-fever, Oropouche and Sandfly fever Naples.
Throughout the year, the collection receives virus deposits. The depositing process is free and simple. The quality of every virus material received in the collection is assessed through a series of tests to confirm viability and absence of microbial contamination (including mycoplasmas). Virus identity is confirmed by nucleic acid profiling. Once standards are met, the virus products are made available to the wider scientific community. In addition to facilitating access to viruses and their associated products as well as secure storage for patents, NCPV also undertakes collaborative projects and contract research/developmental work with academic and commercial partners, by providing expertise in the handling of viral pathogens.
A repository for well-characterised, authenticated viruses is a useful resource for the scientific community, aiding in the understanding of circulating viral pathogens and providing access to quality-assured reference reagents for the identification and development of vaccines and therapeutics in the event of global viral outbreaks.
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DENV-captured plasmin enhances mosquito midgut infection and is inhibited by an endogenous Kazal-type inhibitor AaTI
Deciphering how dengue pathogenesis influences vector transmission will improve our understanding on virulence evolution, epidemiology and design of transmission-blocking tools. Here, we demonstrate that addition to blood meal of plasmin, the human fibrinolytic factor, increased permeability and DENV infectivity in mosquito midgut, resulting in higher infection rate and dissemination in whole mosquitoes. Further, we show that a plasmin-selective mosquito Kazal-type protease inhibitor, AaTI reverted this enhanced infection by inhibiting proteolysis. We also determined that DENV or E-protein, plasmin (not plasminogen) and AaTI can interact to form a tripartite complex using biolayer interferometry, suggesting physical interaction between DENV E-protein and kringle domain of plasmin. Our study suggests that (a) DENV recruit plasmin in solution to increase local proteolytic activity in midgut, thus enhancing DENV infection and (b) AaTI can act as a transmission-blocking agent, which could also alleviate hemorrhagic patients. By discovering that dengue pathogenesis can enhance DENV fitness by increasing mosquito infectivity, our results provide the first evidence of mosquito-based evolutionary pressures on dengue virulence in human. We are currently determining the effect of plasmin and AaTI on DENV infection in cellular and mouse models. H/D exchange mass spectrometry is being employed to identify which kringle domain of plasmin is interacting with DENV E-protein. The structure of the complex formed between the kringle domain of plasmin and DENV E-protein and between the catalytic domain of plasmin and AaTI are in progress.
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DENV-captured plasmin enhances mosquito midgut infection and is inhibited by an endogenous Kazal-type inhibitor AaTI
Deciphering how dengue pathogenesis influences vector transmission will improve our understanding on virulence evolution, epidemiology and design of transmission-blocking tools. Here, we demonstrate that addition to blood meal of plasmin, the human fibrinolytic factor, increased permeability and DENV infectivity in mosquito midgut, resulting in higher infection rate and dissemination in whole mosquitoes. Further, we show that a plasmin-selective mosquito Kazal-type protease inhibitor, AaTI reverted this enhanced infection by inhibiting proteolysis. We also determined that DENV or E-protein, plasmin (not plasminogen) and AaTI can interact to form a tripartite complex using biolayer interferometry, suggesting physical interaction between DENV E-protein and kringle domain of plasmin. Our study suggests that (a) DENV recruit plasmin in solution to increase local proteolytic activity in midgut, thus enhancing DENV infection and (b) AaTI can act as a transmission-blocking agent, which could also alleviate hemorrhagic patients. By discovering that dengue pathogenesis can enhance DENV fitness by increasing mosquito infectivity, our results provide the first evidence of mosquito-based evolutionary pressures on dengue virulence in human. We are currently determining the effect of plasmin and AaTI on DENV infection in cellular and mouse models. H/D exchange mass spectrometry is being employed to identify which kringle domain of plasmin is interacting with DENV E-protein. The structure of the complex formed between the kringle domain of plasmin and DENV E-protein and between the catalytic domain of plasmin and AaTI are in progress.
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Glucose regulated protein 78 (GRP78) interacts with Zika virus envelope and is required for a productive infection
Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated.
To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection.
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Glucose regulated protein 78 (GRP78) interacts with Zika virus envelope and is required for a productive infection
Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated.
To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection.
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Pan-antivirals to combat re-emerging alphaviruses
Mosquito-transmitted alphaviruses are distributed globally and include human pathogens that can cause severe long-term arthritogenic or neurological complications. There are currently no market-approved antivirals or vaccines to treat or prevent these infections. Outbreaks are difficult to predict as they can emerge spontaneously in susceptible human populations. Furthermore, it is challenging to determine the causative pathogen due to great similarities in clinical features of alphavirus-associated diseases. Therefore, drugs with broad-spectrum anti-alphavirus activity could serve as a fast first-line therapy in case of an outbreak. We have performed high-throughput screenings of broad-chemical space libraries and identified a series of small molecules with antiviral activity against different chikungunya virus lineages. Interestingly, we demonstrated that this series exerts broad-spectrum anti-alphavirus activity against a range of arthritogenic alphaviruses. Time-of-addition studies showed that this series has in vitro antiviral activity early in the viral RNA replication stage. Furthermore, these molecules were not cross-resistant with favipiravir-resistant chikungunya virus, a compound that inhibits the viral RNA polymerase. To investigate the mechanism of action in more detail, in vitro resistance selection is currently ongoing. Elucidation of the specific antiviral target of this newly identified series could reveal a new target in the alphavirus proteome, which could be valuable for the development of broad-spectrum anti-alphavirus drugs.
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Pan-antivirals to combat re-emerging alphaviruses
Mosquito-transmitted alphaviruses are distributed globally and include human pathogens that can cause severe long-term arthritogenic or neurological complications. There are currently no market-approved antivirals or vaccines to treat or prevent these infections. Outbreaks are difficult to predict as they can emerge spontaneously in susceptible human populations. Furthermore, it is challenging to determine the causative pathogen due to great similarities in clinical features of alphavirus-associated diseases. Therefore, drugs with broad-spectrum anti-alphavirus activity could serve as a fast first-line therapy in case of an outbreak. We have performed high-throughput screenings of broad-chemical space libraries and identified a series of small molecules with antiviral activity against different chikungunya virus lineages. Interestingly, we demonstrated that this series exerts broad-spectrum anti-alphavirus activity against a range of arthritogenic alphaviruses. Time-of-addition studies showed that this series has in vitro antiviral activity early in the viral RNA replication stage. Furthermore, these molecules were not cross-resistant with favipiravir-resistant chikungunya virus, a compound that inhibits the viral RNA polymerase. To investigate the mechanism of action in more detail, in vitro resistance selection is currently ongoing. Elucidation of the specific antiviral target of this newly identified series could reveal a new target in the alphavirus proteome, which could be valuable for the development of broad-spectrum anti-alphavirus drugs.
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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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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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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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