- Volume 1, Issue 10, 2019

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.