- 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.