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Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogen

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogen

In the complex world of disease transmission, mosquitoes often take centre stage as unwitting actors in the spread of viruses that threaten human and animal health. Imagine this: a tiny mosquito, often overlooked in the grand scheme of nature, carrying within it the potential to cause widespread disease, earning it the title of the world's deadliest animal. This doctoral thesis explores the captivating realm of mosquito-borne viruses, shedding light on their emergence, transmission dynamics, and evolutionary development.Arboviruses such as Japanese encephalitis virus (JEV), Usutu virus (USUV) and chikungunya virus (CHIKV) pose a significant global health problem given their increasing spread, particularly in growing urban areas, and amid today’s climate variations and change. It is important to understand the transmission dynamics of these viruses. In particular, USUV, which is transmitted by Culex mosquitoes, is increasingly being reported in Europe. This warrants an understanding of the vector competence of different European mosquito species to predict and manage potential outbreaks. Similarly, JEV is widespread in Asia and, given the climatic similarities, warrants an investigation into the vector competence of European mosquito species. CHIKV with its different lineages emphasises the need to understand its evolutionary history in order to predict and contain future outbreaks.This work has three objectives. To determine the abundance of JEV vector mosquitoes in Hanoi, Vietnam, to assess the vector competence of Swedish mosquitoes for USUV and JEV, and to analyse the factors that contributed to the 2018 CHIKV outbreak in Thailand. Field collections in Vietnam analysed Culex populations and correlated seasonal abundance with disease incidence. Vector competence experiments revealed USUV transmission potential and emphasised the inability of JEV transmission by Swedish Cx. pipiens. A genomic analysis of CHIKV traced its evolutionary development and provided insigh

Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average Mn ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.

In the complex world of disease transmission, mosquitoes often take centre stage as unwitting actors in the spread of viruses that threaten human and animal health. Imagine this: a tiny mosquito, often overlooked in the grand scheme of nature, carrying within it the potential to cause widespread disease, earning it the title of the world's deadliest animal. This doctoral thesis explores the captivating realm of mosquito-borne viruses, shedding light on their emergence, transmission dynamics, and evolutionary development.Arboviruses such as Japanese encephalitis virus (JEV), Usutu virus (USUV) and chikungunya virus (CHIKV) pose a significant global health problem given their increasing spread, particularly in growing urban areas, and amid today’s climate variations and change. It is important to understand the transmission dynamics of these viruses. In particular, USUV, which is transmitted by Culex mosquitoes, is increasingly being reported in Europe. This warrants an understanding of the vector competence of different European mosquito species to predict and manage potential outbreaks. Similarly, JEV is widespread in Asia and, given the climatic similarities, warrants an investigation into the vector competence of European mosquito species. CHIKV with its different lineages emphasises the need to understand its evolutionary history in order to predict and contain future outbreaks.This work has three objectives. To determine the abundance of JEV vector mosquitoes in Hanoi, Vietnam, to assess the vector competence of Swedish mosquitoes for USUV and JEV, and to analyse the factors that contributed to the 2018 CHIKV outbreak in Thailand. Field collections in Vietnam analysed Culex populations and correlated seasonal abundance with disease incidence. Vector competence experiments revealed USUV transmission potential and emphasised the inability of JEV transmission by Swedish Cx. pipiens. A genomic analysis of CHIKV traced its evolutionary development and provided insigh

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogen

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogen

In the complex world of disease transmission, mosquitoes often take centre stage as unwitting actors in the spread of viruses that threaten human and animal health. Imagine this: a tiny mosquito, often overlooked in the grand scheme of nature, carrying within it the potential to cause widespread disease, earning it the title of the world's deadliest animal. This doctoral thesis explores the captivating realm of mosquito-borne viruses, shedding light on their emergence, transmission dynamics, and evolutionary development.Arboviruses such as Japanese encephalitis virus (JEV), Usutu virus (USUV) and chikungunya virus (CHIKV) pose a significant global health problem given their increasing spread, particularly in growing urban areas, and amid today’s climate variations and change. It is important to understand the transmission dynamics of these viruses. In particular, USUV, which is transmitted by Culex mosquitoes, is increasingly being reported in Europe. This warrants an understanding of the vector competence of different European mosquito species to predict and manage potential outbreaks. Similarly, JEV is widespread in Asia and, given the climatic similarities, warrants an investigation into the vector competence of European mosquito species. CHIKV with its different lineages emphasises the need to understand its evolutionary history in order to predict and contain future outbreaks.This work has three objectives. To determine the abundance of JEV vector mosquitoes in Hanoi, Vietnam, to assess the vector competence of Swedish mosquitoes for USUV and JEV, and to analyse the factors that contributed to the 2018 CHIKV outbreak in Thailand. Field collections in Vietnam analysed Culex populations and correlated seasonal abundance with disease incidence. Vector competence experiments revealed USUV transmission potential and emphasised the inability of JEV transmission by Swedish Cx. pipiens. A genomic analysis of CHIKV traced its evolutionary development and provided insigh

In the complex world of disease transmission, mosquitoes often take centre stage as unwitting actors in the spread of viruses that threaten human and animal health. Imagine this: a tiny mosquito, often overlooked in the grand scheme of nature, carrying within it the potential to cause widespread disease, earning it the title of the world's deadliest animal. This doctoral thesis explores the captivating realm of mosquito-borne viruses, shedding light on their emergence, transmission dynamics, and evolutionary development.Arboviruses such as Japanese encephalitis virus (JEV), Usutu virus (USUV) and chikungunya virus (CHIKV) pose a significant global health problem given their increasing spread, particularly in growing urban areas, and amid today’s climate variations and change. It is important to understand the transmission dynamics of these viruses. In particular, USUV, which is transmitted by Culex mosquitoes, is increasingly being reported in Europe. This warrants an understanding of the vector competence of different European mosquito species to predict and manage potential outbreaks. Similarly, JEV is widespread in Asia and, given the climatic similarities, warrants an investigation into the vector competence of European mosquito species. CHIKV with its different lineages emphasises the need to understand its evolutionary history in order to predict and contain future outbreaks.This work has three objectives. To determine the abundance of JEV vector mosquitoes in Hanoi, Vietnam, to assess the vector competence of Swedish mosquitoes for USUV and JEV, and to analyse the factors that contributed to the 2018 CHIKV outbreak in Thailand. Field collections in Vietnam analysed Culex populations and correlated seasonal abundance with disease incidence. Vector competence experiments revealed USUV transmission potential and emphasised the inability of JEV transmission by Swedish Cx. pipiens. A genomic analysis of CHIKV traced its evolutionary development and provided insigh

Intraspecies (homologous) phylogenetic incongruence, or 'tree conflict' between different loci within the same genome of mosquito-borne flaviviruses (MBFV), was first identified in dengue virus (DENV) and subsequently in Japanese encephalitis virus (JEV), St Louis encephalitis virus, and Zika virus (ZIKV). Recently, the first evidence of phylogenetic incongruence between interspecific members of the MBFV was reported in ZIKV and its close relative, Spondweni virus. Uniquely, these hybrid proteomes were derived from four incongruent trees involving an Aedes-associated DENV node (1 tree) and three different Culex-associated flavivirus nodes (3 trees). This analysis has now been extended across a wider spectrum of viruses within the MBFV lineage targeting the breakpoints between phylogenetic incongruent loci originally identified in ZIKV. Interspecies phylogenetic incongruence at these breakpoints was identified in 10 of 50 viruses within the MBFV lineage, representing emergent Aedes and Culex-associated viruses including JEV, West Nile virus, yellow fever virus, and insect-specific viruses. Thus, interspecies phylogenetic incongruence is widespread amongst the flaviviruses and is robustly associated with the specific breakpoints that coincide with the interspecific phylogenetic incongruence previously identified, inferring they are 'hotspots'. The incongruence amongst the emergent MBFV group was restricted to viruses within their respective associated epidemiological boundaries. This MBFV group was RY-coded at the third codon position ('wobble codon') to remove transition saturation. The resulting 'wobble codon' trees presented a single topology for the entire genome that lacked any robust evidence of phylogenetic incongruence between loci. Phylogenetic interspecific incongruence was therefore observed for exactly the same loci between amino acid and the RY-coded 'wobble codon' alignments and this incongruence represented either a major part, or the entire genomes. Ma

Intraspecies (homologous) phylogenetic incongruence, or 'tree conflict' between different loci within the same genome of mosquito-borne flaviviruses (MBFV), was first identified in dengue virus (DENV) and subsequently in Japanese encephalitis virus (JEV), St Louis encephalitis virus, and Zika virus (ZIKV). Recently, the first evidence of phylogenetic incongruence between interspecific members of the MBFV was reported in ZIKV and its close relative, Spondweni virus. Uniquely, these hybrid proteomes were derived from four incongruent trees involving an Aedes-associated DENV node (1 tree) and three different Culex-associated flavivirus nodes (3 trees). This analysis has now been extended across a wider spectrum of viruses within the MBFV lineage targeting the breakpoints between phylogenetic incongruent loci originally identified in ZIKV. Interspecies phylogenetic incongruence at these breakpoints was identified in 10 of 50 viruses within the MBFV lineage, representing emergent Aedes and Culex-associated viruses including JEV, West Nile virus, yellow fever virus, and insect-specific viruses. Thus, interspecies phylogenetic incongruence is widespread amongst the flaviviruses and is robustly associated with the specific breakpoints that coincide with the interspecific phylogenetic incongruence previously identified, inferring they are 'hotspots'. The incongruence amongst the emergent MBFV group was restricted to viruses within their respective associated epidemiological boundaries. This MBFV group was RY-coded at the third codon position ('wobble codon') to remove transition saturation. The resulting 'wobble codon' trees presented a single topology for the entire genome that lacked any robust evidence of phylogenetic incongruence between loci. Phylogenetic interspecific incongruence was therefore observed for exactly the same loci between amino acid and the RY-coded 'wobble codon' alignments and this incongruence represented either a major part, or the entire genomes. Ma

Intraspecies (homologous) phylogenetic incongruence, or 'tree conflict' between different loci within the same genome of mosquito-borne flaviviruses (MBFV), was first identified in dengue virus (DENV) and subsequently in Japanese encephalitis virus (JEV), St Louis encephalitis virus, and Zika virus (ZIKV). Recently, the first evidence of phylogenetic incongruence between interspecific members of the MBFV was reported in ZIKV and its close relative, Spondweni virus. Uniquely, these hybrid proteomes were derived from four incongruent trees involving an Aedes-associated DENV node (1 tree) and three different Culex-associated flavivirus nodes (3 trees). This analysis has now been extended across a wider spectrum of viruses within the MBFV lineage targeting the breakpoints between phylogenetic incongruent loci originally identified in ZIKV. Interspecies phylogenetic incongruence at these breakpoints was identified in 10 of 50 viruses within the MBFV lineage, representing emergent Aedes and Culex-associated viruses including JEV, West Nile virus, yellow fever virus, and insect-specific viruses. Thus, interspecies phylogenetic incongruence is widespread amongst the flaviviruses and is robustly associated with the specific breakpoints that coincide with the interspecific phylogenetic incongruence previously identified, inferring they are 'hotspots'. The incongruence amongst the emergent MBFV group was restricted to viruses within their respective associated epidemiological boundaries. This MBFV group was RY-coded at the third codon position ('wobble codon') to remove transition saturation. The resulting 'wobble codon' trees presented a single topology for the entire genome that lacked any robust evidence of phylogenetic incongruence between loci. Phylogenetic interspecific incongruence was therefore observed for exactly the same loci between amino acid and the RY-coded 'wobble codon' alignments and this incongruence represented either a major part, or the entire genomes. Ma

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Intraspecies (homologous) phylogenetic incongruence, or 'tree conflict' between different loci within the same genome of mosquito-borne flaviviruses (MBFV), was first identified in dengue virus (DENV) and subsequently in Japanese encephalitis virus (JEV), St Louis encephalitis virus, and Zika virus (ZIKV). Recently, the first evidence of phylogenetic incongruence between interspecific members of the MBFV was reported in ZIKV and its close relative, Spondweni virus. Uniquely, these hybrid proteomes were derived from four incongruent trees involving an Aedes-associated DENV node (1 tree) and three different Culex-associated flavivirus nodes (3 trees). This analysis has now been extended across a wider spectrum of viruses within the MBFV lineage targeting the breakpoints between phylogenetic incongruent loci originally identified in ZIKV. Interspecies phylogenetic incongruence at these breakpoints was identified in 10 of 50 viruses within the MBFV lineage, representing emergent Aedes and Culex-associated viruses including JEV, West Nile virus, yellow fever virus, and insect-specific viruses. Thus, interspecies phylogenetic incongruence is widespread amongst the flaviviruses and is robustly associated with the specific breakpoints that coincide with the interspecific phylogenetic incongruence previously identified, inferring they are 'hotspots'. The incongruence amongst the emergent MBFV group was restricted to viruses within their respective associated epidemiological boundaries. This MBFV group was RY-coded at the third codon position ('wobble codon') to remove transition saturation. The resulting 'wobble codon' trees presented a single topology for the entire genome that lacked any robust evidence of phylogenetic incongruence between loci. Phylogenetic interspecific incongruence was therefore observed for exactly the same loci between amino acid and the RY-coded 'wobble codon' alignments and this incongruence represented either a major part, or the entire genomes. Ma

Intraspecies (homologous) phylogenetic incongruence, or 'tree conflict' between different loci within the same genome of mosquito-borne flaviviruses (MBFV), was first identified in dengue virus (DENV) and subsequently in Japanese encephalitis virus (JEV), St Louis encephalitis virus, and Zika virus (ZIKV). Recently, the first evidence of phylogenetic incongruence between interspecific members of the MBFV was reported in ZIKV and its close relative, Spondweni virus. Uniquely, these hybrid proteomes were derived from four incongruent trees involving an Aedes-associated DENV node (1 tree) and three different Culex-associated flavivirus nodes (3 trees). This analysis has now been extended across a wider spectrum of viruses within the MBFV lineage targeting the breakpoints between phylogenetic incongruent loci originally identified in ZIKV. Interspecies phylogenetic incongruence at these breakpoints was identified in 10 of 50 viruses within the MBFV lineage, representing emergent Aedes and Culex-associated viruses including JEV, West Nile virus, yellow fever virus, and insect-specific viruses. Thus, interspecies phylogenetic incongruence is widespread amongst the flaviviruses and is robustly associated with the specific breakpoints that coincide with the interspecific phylogenetic incongruence previously identified, inferring they are 'hotspots'. The incongruence amongst the emergent MBFV group was restricted to viruses within their respective associated epidemiological boundaries. This MBFV group was RY-coded at the third codon position ('wobble codon') to remove transition saturation. The resulting 'wobble codon' trees presented a single topology for the entire genome that lacked any robust evidence of phylogenetic incongruence between loci. Phylogenetic interspecific incongruence was therefore observed for exactly the same loci between amino acid and the RY-coded 'wobble codon' alignments and this incongruence represented either a major part, or the entire genomes. Ma

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Vector-borne diseases are responsible for more than 20% of the infectious diseases worldwide. The prevalence of arboviruses transmit diseases to humans in Sudan has not been investigated. Mosquito-borne viral diseases increase globally incidence, including the Sudan. Frequent unknown fever outbreaks have been reported in eastern region, Sudan. However, diagnosis was based exclusively on clinical signs and symptoms without confirmatory laboratory investigations. However, for accurate detection of these viruses in outbreaks, molecular technique is considered. The objective of this study was to determine the prevalence of six arboviruses in the Kassala state of east Sudan during unknown fever outbreak. A cross sectional hospital-based study was conducted in the Kassala, Teaching Hospital. Blood samples from 119 patients suffering from unknown fever were used for screening of six arboviruses, hepatitis E virus and malarial using molecular techniques and serology. The overall arboviruses seroprevelance was 61.3% (73/119). The highest positivity rate was 73.1% (52/73) chikungunya virus; 29 males and 20 females patients were chikungunya positive. Other arboviruses were circulating in low rate 20.5% (15/73), and 6.8% (5/73) for sindbis and rift valley fever viruses respectively. Hepatitis E virus was negative in all cases and malaria positivity rate 13.4% (16/119). The prevalence of arboviruses among unknown fever patients present to Kassala teaching hospital of eastern region in Sudan is significantly high (61.3%). The chikungunya virus is the predominant causative agent of arboviruses. Molecular techniques such as PCR are important for accurate and rapid diagnosis of this viral outbreak.

Vector-borne diseases are responsible for more than 20% of the infectious diseases worldwide. The prevalence of arboviruses transmit diseases to humans in Sudan has not been investigated. Mosquito-borne viral diseases increase globally incidence, including the Sudan. Frequent unknown fever outbreaks have been reported in eastern region, Sudan. However, diagnosis was based exclusively on clinical signs and symptoms without confirmatory laboratory investigations. However, for accurate detection of these viruses in outbreaks, molecular technique is considered. The objective of this study was to determine the prevalence of six arboviruses in the Kassala state of east Sudan during unknown fever outbreak. A cross sectional hospital-based study was conducted in the Kassala, Teaching Hospital. Blood samples from 119 patients suffering from unknown fever were used for screening of six arboviruses, hepatitis E virus and malarial using molecular techniques and serology. The overall arboviruses seroprevelance was 61.3% (73/119). The highest positivity rate was 73.1% (52/73) chikungunya virus; 29 males and 20 females patients were chikungunya positive. Other arboviruses were circulating in low rate 20.5% (15/73), and 6.8% (5/73) for sindbis and rift valley fever viruses respectively. Hepatitis E virus was negative in all cases and malaria positivity rate 13.4% (16/119). The prevalence of arboviruses among unknown fever patients present to Kassala teaching hospital of eastern region in Sudan is significantly high (61.3%). The chikungunya virus is the predominant causative agent of arboviruses. Molecular techniques such as PCR are important for accurate and rapid diagnosis of this viral outbreak.

Vector-borne diseases are responsible for more than 20% of the infectious diseases worldwide. The prevalence of arboviruses transmit diseases to humans in Sudan has not been investigated. Mosquito-borne viral diseases increase globally incidence, including the Sudan. Frequent unknown fever outbreaks have been reported in eastern region, Sudan. However, diagnosis was based exclusively on clinical signs and symptoms without confirmatory laboratory investigations. However, for accurate detection of these viruses in outbreaks, molecular technique is considered. The objective of this study was to determine the prevalence of six arboviruses in the Kassala state of east Sudan during unknown fever outbreak. A cross sectional hospital-based study was conducted in the Kassala, Teaching Hospital. Blood samples from 119 patients suffering from unknown fever were used for screening of six arboviruses, hepatitis E virus and malarial using molecular techniques and serology. The overall arboviruses seroprevelance was 61.3% (73/119). The highest positivity rate was 73.1% (52/73) chikungunya virus; 29 males and 20 females patients were chikungunya positive. Other arboviruses were circulating in low rate 20.5% (15/73), and 6.8% (5/73) for sindbis and rift valley fever viruses respectively. Hepatitis E virus was negative in all cases and malaria positivity rate 13.4% (16/119). The prevalence of arboviruses among unknown fever patients present to Kassala teaching hospital of eastern region in Sudan is significantly high (61.3%). The chikungunya virus is the predominant causative agent of arboviruses. Molecular techniques such as PCR are important for accurate and rapid diagnosis of this viral outbreak.

Background: The European Commission (EC) Horizon 2020 (H2020)-funded ZIKAlliance Consortium designed a multicentre study including pregnant women (PW), children (CH) and natural history (NH) cohorts. Clinical sites were selected over a wide geographic range within Latin America and the Caribbean, taking into account the dynamic course of the ZIKV epidemic. Methods: Recruitment to the PW cohort will take place in antenatal care clinics. PW will be enrolled regardless of symptoms and followed over the course of pregnancy, approximately every 4 weeks. PW will be revisited at delivery (or after miscarriage/abortion) to assess birth outcomes, including microcephaly and other congenital abnormalities according to the evolving definition of congenital Zika syndrome (CZS). After birth, children will be followed for 2 years in the CH cohort. Follow-up visits are scheduled at ages 1-3, 4-6, 12, and 24 months to assess neurocognitive and developmental milestones. In addition, a NH cohort for the characterization of symptomatic rash/fever illness was designed, including follow-up to capture persisting health problems. Blood, urine, and other biological materials will be collected, and tested for ZIKV and other relevant arboviral diseases (dengue, chikungunya, yellow fever) using RT-PCR or serological methods. A virtual, decentralized biobank will be created. Reciprocal clinical monitoring has been established between partner sites. Substudies of ZIKV seroprevalence, transmission

Vector-borne diseases are responsible for more than 20% of the infectious diseases worldwide. The prevalence of arboviruses transmit diseases to humans in Sudan has not been investigated. Mosquito-borne viral diseases increase globally incidence, including the Sudan. Frequent unknown fever outbreaks have been reported in eastern region, Sudan. However, diagnosis was based exclusively on clinical signs and symptoms without confirmatory laboratory investigations. However, for accurate detection of these viruses in outbreaks, molecular technique is considered. The objective of this study was to determine the prevalence of six arboviruses in the Kassala state of east Sudan during unknown fever outbreak. A cross sectional hospital-based study was conducted in the Kassala, Teaching Hospital. Blood samples from 119 patients suffering from unknown fever were used for screening of six arboviruses, hepatitis E virus and malarial using molecular techniques and serology. The overall arboviruses seroprevelance was 61.3% (73/119). The highest positivity rate was 73.1% (52/73) chikungunya virus; 29 males and 20 females patients were chikungunya positive. Other arboviruses were circulating in low rate 20.5% (15/73), and 6.8% (5/73) for sindbis and rift valley fever viruses respectively. Hepatitis E virus was negative in all cases and malaria positivity rate 13.4% (16/119). The prevalence of arboviruses among unknown fever patients present to Kassala teaching hospital of eastern region in Sudan is significantly high (61.3%). The chikungunya virus is the predominant causative agent of arboviruses. Molecular techniques such as PCR are important for accurate and rapid diagnosis of this viral outbreak.

Mosquitoes are vectors of arboviruses affecting animal and human health. Arboviruses circulate primarily within an enzootic cycle and recurrent spillovers contribute to the emergence of human-adapted viruses able to initiate an urban cycle involving anthropophilic mosquitoes. The increasing volume of travel and trade offers multiple opportunities for arbovirus introduction in new regions. This scenario has been exemplified recently with the Zika pandemic. To incriminate a mosquito as vector of a pathogen, several criteria are required such as the detection of natural infections in mosquitoes. In this study, we used a high-throughput chip based on the BioMark™ Dynamic arrays system capable of detecting 64 arboviruses in a single experiment. A total of 17,958 mosquitoes collected in Zika-endemic/epidemic countries (Brazil, French Guiana, Guadeloupe, Suriname, Senegal, and Cambodia) were analyzed. Here we show that this new tool can detect endemic and epidemic viruses in different mosquito species in an epidemic context. Thus, this fast and low-cost method can be suggested as a novel epidemiological surveillance tool to identify circulating arboviruses.

Vector-borne diseases are responsible for more than 20% of the infectious diseases worldwide. The prevalence of arboviruses transmit diseases to humans in Sudan has not been investigated. Mosquito-borne viral diseases increase globally incidence, including the Sudan. Frequent unknown fever outbreaks have been reported in eastern region, Sudan. However, diagnosis was based exclusively on clinical signs and symptoms without confirmatory laboratory investigations. However, for accurate detection of these viruses in outbreaks, molecular technique is considered. The objective of this study was to determine the prevalence of six arboviruses in the Kassala state of east Sudan during unknown fever outbreak. A cross sectional hospital-based study was conducted in the Kassala, Teaching Hospital. Blood samples from 119 patients suffering from unknown fever were used for screening of six arboviruses, hepatitis E virus and malarial using molecular techniques and serology. The overall arboviruses seroprevelance was 61.3% (73/119). The highest positivity rate was 73.1% (52/73) chikungunya virus; 29 males and 20 females patients were chikungunya positive. Other arboviruses were circulating in low rate 20.5% (15/73), and 6.8% (5/73) for sindbis and rift valley fever viruses respectively. Hepatitis E virus was negative in all cases and malaria positivity rate 13.4% (16/119). The prevalence of arboviruses among unknown fever patients present to Kassala teaching hospital of eastern region in Sudan is significantly high (61.3%). The chikungunya virus is the predominant causative agent of arboviruses. Molecular techniques such as PCR are important for accurate and rapid diagnosis of this viral outbreak.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medical Science, Griffith Health, Many Flaviviruses are mosquito-borne viruses that can causes disease in humans and animals including Dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV). These viruses are highly pathogenic to humans causing extensive morbidity and mortality and most importantly, their recent re-emergence and global spread is a current growing public health issue. Currently, DENV alone causes an approximate 50 million infection cases annually and WNV infections have been reported in the five continents. Despite the existence of licensed vaccines for YFV, JEV and TBEV, a major challenge remains given there are no specific and effective antiviral therapies against flaviviral infections. Considering the homologies between flaviviral replication strategies, it may be possible to identify broad-spectrum compounds with both prophylactic and therapeutic activity against different flavivirus strains. Because the steps involved in compound discovery to their clinical use are protracted and expensive, studies should include testing existing clinically approved drugs as antivirals. Melaleuca alternifolia extracts are good candidates to be explored for novel uses as they have been extensively studied and their composition, chemistry, safety and toxicity are well documented. Melaleluca alternifolia concentrate (MAC) is obtained from the further purification of Tea Tree Oil (TTO) and is a new product exhibiting promising antimicrobial and anti-cancer activities.

Many Flaviviruses are mosquito-borne viruses that can causes disease in humans and animals including Dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV). These viruses are highly pathogenic to humans causing extensive morbidity and mortality and most importantly, their recent re-emergence and global spread is a current growing public health issue. Currently, DENV alone causes an approximate 50 million infection cases annually and WNV infections have been reported in the five continents. Despite the existence of licensed vaccines for YFV, JEV and TBEV, a major challenge remains given there are no specific and effective antiviral therapies against flaviviral infections. Considering the homologies between flaviviral replication strategies, it may be possible to identify broad-spectrum compounds with both prophylactic and therapeutic activity against different flavivirus strains. Because the steps involved in compound discovery to their clinical use are protracted and expensive, studies should include testing existing clinically approved drugs as antivirals. Melaleuca alternifolia extracts are good candidates to be explored for novel uses as they have been extensively studied and their composition, chemistry, safety and toxicity are well documented. Melaleluca alternifolia concentrate (MAC) is obtained from the further purification of Tea Tree Oil (TTO) and is a new product exhibiting promising antimicrobial and anti-cancer activities., Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Medical Science, Griffith Health, Full Text