Access Microbiology - Volume 1, Issue 10, 2019

In Europe, tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are two flaviviruses both transmitted by the tick Ixodes ricinus. While the mechanisms of viral replication and transmission in this vector are incompletely understood, they are presumed to be largely governed by protein-protein interactions established between viruses and cells.

To elucidate the molecular determinants involved in vector competence, we have mapped the network of protein-protein interactions established between viral proteins of both TBEV and LIV and tick proteins encoded by a cDNA library of I. ricinus, by using yeast two-hybrid methodology. Twenty-two cellular partners from I. ricinus have been identified and all evidenced to interact with both viruses. Upon functional annotation, some of these tick proteins seem to be involved in such biological processes as the immune response or ribosomal maturation. To gain insight into the role of each tick protein in viral replication, the impact of gene silencing will be assessed by dsRNA knockdown. In parallel, I. ricinus cell lines have been persistently infected with TBEV or LIV and the level of expression of selected antiviral effectors monitored over time, in an effort to characterize the antiviral response of this arthropod.

This work represents the first description of the protein-protein interaction network for TBEV, LIV and I. ricinus. Certain tick partners may well represent molecular determinants of vector competence of I. ricinus for TBEV and LIV and potentially other flaviviruses, which we will ascertain by in vivo silencing of selected tick partners.

In Europe, tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are two flaviviruses both transmitted by the tick Ixodes ricinus. While the mechanisms of viral replication and transmission in this vector are incompletely understood, they are presumed to be largely governed by protein-protein interactions established between viruses and cells.

To elucidate the molecular determinants involved in vector competence, we have mapped the network of protein-protein interactions established between viral proteins of both TBEV and LIV and tick proteins encoded by a cDNA library of I. ricinus, by using yeast two-hybrid methodology. Twenty-two cellular partners from I. ricinus have been identified and all evidenced to interact with both viruses. Upon functional annotation, some of these tick proteins seem to be involved in such biological processes as the immune response or ribosomal maturation. To gain insight into the role of each tick protein in viral replication, the impact of gene silencing will be assessed by dsRNA knockdown. In parallel, I. ricinus cell lines have been persistently infected with TBEV or LIV and the level of expression of selected antiviral effectors monitored over time, in an effort to characterize the antiviral response of this arthropod.

This work represents the first description of the protein-protein interaction network for TBEV, LIV and I. ricinus. Certain tick partners may well represent molecular determinants of vector competence of I. ricinus for TBEV and LIV and potentially other flaviviruses, which we will ascertain by in vivo silencing of selected tick partners.

Background. The continuous use of insecticides for mosquito-borne disease control can cause insecticide resistance, and if left unchecked, this could lead to a substantial increase in disease incidence. The aims of this study are to monitor insecticide insecticides against Malathion and Cypermethrin and identifying the mechanisms underlying the resistance in the area of study.

Method. This is a descriptive study located in Banyumas Regency. Aedes sp. mosquitoes werecollected from three endemic areas (Arcawinangun, Karangpucung, and Purwanegara) by the ovitrap installation to 100 houses each village (total 300 houses). Filial 1 of Aedes were tested their insecticide resistance to Malathion and Cypermethrin by susceptibility test, biochemical assay and molecular by PCR.

Results. The results of the susceptibility test showed the average percentage of mosquito mortality from three villages was 30,67% which were included in the resistance category. However, the results of the biochemical assay showed that 70% of mosquitoes are still very susceptible (AV<0,7). Molecular tests are underway and the results are likely to be obtained in August 2019

Conclusion. The population of Aedes sp. in the study area has been resistant to malathion and cypermethrin, and the mechanism underlying this resistance was not based on a biochemical mechanism. It is necessary to rotate the use of insecticide active substances in DHF vector control by selecting insecticides that have a different mode of action.

Background. The continuous use of insecticides for mosquito-borne disease control can cause insecticide resistance, and if left unchecked, this could lead to a substantial increase in disease incidence. The aims of this study are to monitor insecticide insecticides against Malathion and Cypermethrin and identifying the mechanisms underlying the resistance in the area of study.

Method. This is a descriptive study located in Banyumas Regency. Aedes sp. mosquitoes werecollected from three endemic areas (Arcawinangun, Karangpucung, and Purwanegara) by the ovitrap installation to 100 houses each village (total 300 houses). Filial 1 of Aedes were tested their insecticide resistance to Malathion and Cypermethrin by susceptibility test, biochemical assay and molecular by PCR.

Results. The results of the susceptibility test showed the average percentage of mosquito mortality from three villages was 30,67% which were included in the resistance category. However, the results of the biochemical assay showed that 70% of mosquitoes are still very susceptible (AV<0,7). Molecular tests are underway and the results are likely to be obtained in August 2019

Conclusion. The population of Aedes sp. in the study area has been resistant to malathion and cypermethrin, and the mechanism underlying this resistance was not based on a biochemical mechanism. It is necessary to rotate the use of insecticide active substances in DHF vector control by selecting insecticides that have a different mode of action.

Yellow fever mosquitoes (Aedes aegypti) and Asian tiger mosquitoes (Aedes albopictus) are the primary vectors of dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV). These viruses are transmitted to humans through mosquito saliva, making the vector salivary gland (SG) a critical tissue to identify transmission-blocking targets. We examined gene expression in infected SGs for both vector species and for three different virus infections. Aedes aegypti SGs were infected separately with DENV, ZIKV and CHIKV, and Ae. albopictus with CHIKV. RNA-sequencing identified differentially expressed coding and long non-coding RNAs (lncRNAs). Differentially expressed genes determined from genome annotations were greater in number and functional diversity in comparison to differentially expressed transcripts from de novo transcriptome assemblies. Salivary protein transcripts were the most abundant, but were downregulated in all three virus infections. Commonly upregulated genes were associated with apoptosis, cytoskeletal proteins, replication/transcription/translation, redox/stress and immunity. An enrichment of upregulated genes related to apoptosis were observed in CHIKV infection in comparison to DENV and ZIKV infections. Upregulation of serine proteases and other genes associated with immunity and cellular stress responses (cytochrome P450 genes) varied between vectors. There were also immune response commonalities between vectors, for instance RNA-interference was observed to be a non-specific antiviral defense. The number of lncRNA transcripts differentially expressed were few and none were common to all infections, likely having minor roles, unlike the lncRNA antiviral effects proposed for mosquito midgut. Determining common infection patterns for different viruses and vectors has applications in refractory vector engineering.

Yellow fever mosquitoes (Aedes aegypti) and Asian tiger mosquitoes (Aedes albopictus) are the primary vectors of dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV). These viruses are transmitted to humans through mosquito saliva, making the vector salivary gland (SG) a critical tissue to identify transmission-blocking targets. We examined gene expression in infected SGs for both vector species and for three different virus infections. Aedes aegypti SGs were infected separately with DENV, ZIKV and CHIKV, and Ae. albopictus with CHIKV. RNA-sequencing identified differentially expressed coding and long non-coding RNAs (lncRNAs). Differentially expressed genes determined from genome annotations were greater in number and functional diversity in comparison to differentially expressed transcripts from de novo transcriptome assemblies. Salivary protein transcripts were the most abundant, but were downregulated in all three virus infections. Commonly upregulated genes were associated with apoptosis, cytoskeletal proteins, replication/transcription/translation, redox/stress and immunity. An enrichment of upregulated genes related to apoptosis were observed in CHIKV infection in comparison to DENV and ZIKV infections. Upregulation of serine proteases and other genes associated with immunity and cellular stress responses (cytochrome P450 genes) varied between vectors. There were also immune response commonalities between vectors, for instance RNA-interference was observed to be a non-specific antiviral defense. The number of lncRNA transcripts differentially expressed were few and none were common to all infections, likely having minor roles, unlike the lncRNA antiviral effects proposed for mosquito midgut. Determining common infection patterns for different viruses and vectors has applications in refractory vector engineering.

Tick-borne encephalitis virus (TBEV; Flaviviridae) can cause serious infections in humans which may result in encephalitis/meningoencephalitis. It has been previously reported that TBEV infects both, neurons and astrocytes, however, with a different outcome. So far, the principle of this cell type-specific response to TBEV is not fully understood.

In order to gain more insight into this phenomenon, we described new in vitro infection model utilizing human neural stem cells (hNSCs) and two strains of Western European TBEV subtype varying in the pathogenicity - mild Neudoerfl and severe Hypr. In detail, neurons and astrocytes were artificially differentiated from hNSCs and presence of CNS markers was checked. TBEV infection in both cell types was characterised afterwards. As expected, both cell types proved to be susceptible to TBEV infection. Viability was negatively affected only in infected neurons. In order to identify possible effectors responsible for different susceptibility of neurons and astrocytes, the analyses of changes in poly-(A) and small RNA transcriptome upon TBEV infection were performed. Preliminary results from poly-(A) RNA transcriptome revealed that in both cell types mainly interferon-stimulated genes (ISGs) were up-regulated. However, the expression kinetics of particular ISGs varied. In addition, the vast spectrum of long non-coding RNAs was described to be differentially expressed upon infection. Surprisingly, U1 snRNA was found to be the most down-regulated RNA species among almost all infected samples.

Further analyses are in progress in order to get a complete description of virus-induced changes on the transcriptomic level.

Tick-borne encephalitis virus (TBEV; Flaviviridae) can cause serious infections in humans which may result in encephalitis/meningoencephalitis. It has been previously reported that TBEV infects both, neurons and astrocytes, however, with a different outcome. So far, the principle of this cell type-specific response to TBEV is not fully understood.

In order to gain more insight into this phenomenon, we described new in vitro infection model utilizing human neural stem cells (hNSCs) and two strains of Western European TBEV subtype varying in the pathogenicity - mild Neudoerfl and severe Hypr. In detail, neurons and astrocytes were artificially differentiated from hNSCs and presence of CNS markers was checked. TBEV infection in both cell types was characterised afterwards. As expected, both cell types proved to be susceptible to TBEV infection. Viability was negatively affected only in infected neurons. In order to identify possible effectors responsible for different susceptibility of neurons and astrocytes, the analyses of changes in poly-(A) and small RNA transcriptome upon TBEV infection were performed. Preliminary results from poly-(A) RNA transcriptome revealed that in both cell types mainly interferon-stimulated genes (ISGs) were up-regulated. However, the expression kinetics of particular ISGs varied. In addition, the vast spectrum of long non-coding RNAs was described to be differentially expressed upon infection. Surprisingly, U1 snRNA was found to be the most down-regulated RNA species among almost all infected samples.

Further analyses are in progress in order to get a complete description of virus-induced changes on the transcriptomic level.

Tick-borne encephalitis virus (TBEV, Flaviviridae) infection causes severe neurological disease and incapacitates more than 10 000 patients annually in the Eurasian region. Despite extensive studies, some areas of interaction of TBEV with the host cells remain undescribed. Here we investigated the interaction of TBEV and human neural DAOY HTB-186 cells on the transcriptional and translational level.

By labelling of nascent RNA and protein molecules in TBEV-infected DAOY cells, we showed that the virus-induced host translational shut-off. Moreover, TBEV interfered also with the expression of host ribosomal RNAs, in particular with the rRNA species transcribed by RNA polymerase I (18S rRNA, 28S rRNA, and their precursor 45-47S pre-rRNA). Synthesis of host rRNAs is an essential host cell process that is localized in the nucleus, namely nucleoli. By searching for virus factor that could be linked with these effects, we described so far unknown nucleolar localization of TBEV capsid protein C. More importantly, preliminary data from transfection of recombinant C protein led to the reduction in nascent protein synthesis indicating the link between TBEV capsid protein and shut-off phenomena which were described. Furthermore, we identified a potential nuclear localization signal, which seems not to be essential for the shut-down effect.

Taken together we described a brand new type of interaction between TBEV and host neural cells on the transcriptional and translational level and identified viral factor potentially responsible for the observed phenomena. However, further analyses are needed, and the particular mechanism of action remains still elusive.

Tick-borne encephalitis virus (TBEV, Flaviviridae) infection causes severe neurological disease and incapacitates more than 10 000 patients annually in the Eurasian region. Despite extensive studies, some areas of interaction of TBEV with the host cells remain undescribed. Here we investigated the interaction of TBEV and human neural DAOY HTB-186 cells on the transcriptional and translational level.

By labelling of nascent RNA and protein molecules in TBEV-infected DAOY cells, we showed that the virus-induced host translational shut-off. Moreover, TBEV interfered also with the expression of host ribosomal RNAs, in particular with the rRNA species transcribed by RNA polymerase I (18S rRNA, 28S rRNA, and their precursor 45-47S pre-rRNA). Synthesis of host rRNAs is an essential host cell process that is localized in the nucleus, namely nucleoli. By searching for virus factor that could be linked with these effects, we described so far unknown nucleolar localization of TBEV capsid protein C. More importantly, preliminary data from transfection of recombinant C protein led to the reduction in nascent protein synthesis indicating the link between TBEV capsid protein and shut-off phenomena which were described. Furthermore, we identified a potential nuclear localization signal, which seems not to be essential for the shut-down effect.

Taken together we described a brand new type of interaction between TBEV and host neural cells on the transcriptional and translational level and identified viral factor potentially responsible for the observed phenomena. However, further analyses are needed, and the particular mechanism of action remains still elusive.

The development of J93-463-1-16-10 cell culture by sequential adaptation method in serum-free media of Hybridoma-SFM medium and CD Hybridoma medium to produce the monoclonal antibody specific to Japanese encephalitis virus was shown that the viabilities of J93-463-1-16-10 were more than 70%. Then the supernatant from the cell culture was purified after precipitation with ammonium sulfate until the final concentration was 50% in order to separate the IgG. After that, the protein concentrations were measured by using the Bradford assay. Next, the proteins were separated, which made each protein purer with SDS-PAGE method in order to find the molecular weights of the separated proteins. The putative IgG and its heavy chains and light chains were divided in two sizes which were approximate 23-25 kDa and 50-53 kDa respectively. Later, the type of immunoglobulin will be identified by the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) method and its antibody characteristic to Japanese encephalitis virus which will be done further.

In the future, a monoclonal antibody in this research could be used to study the expression of the envelope protein of Japanese encephalitis virus in green cos and red cos, and to develop the vaccine for Japanese encephalitis virus in swine.

Keywords: Japanese encephalitis virus, J93-463-1-16-10, serum-free media, MALDI-TOF

The cryopreservation of cos lettuce (CL) callus by using liquid nitrogen (LN) was further study from the previous research on callus induction and micropropagation of CL. That was studied on seed culture and micropropagation of CL and its embryogenic growth. The result was shown that the most suitable Murashing and Skoog (MS) medium concentration was in 1/2 MS. The medium supplemented with 0.1 mg/l of 6-benzylaminopurine (BAP) and 0.5 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D) gave the best for leaf inducing callus. Then it was brought to study the effects of cryoprotectants with the different percentages of dimethyl sulfoxide (DMSO). The cryopreserved callus by using LN was brought to subculture for the best inducing shoot in MS medium supplemented with 4.0 mg/l of BAP. The results that the best cryoprotectant at the percentage of DMSO at 7.5 gave the highest average number of shoots at 2.1 shoots/culture and the average shoot length was 1.56 cm. The MS medium supplemented with 0.5 mg/l of 1-Naphthaleneacetic acid was the best inducing root. The results that the best cryoprotectant at the percentage of DMSO at 7.5 gave the highest average number of roots at 9.7 roots/culture and the average root length was 1.59 cm. Plant differentiation from callus of CL leaf was transferred to grow in soil and had the same morphology as normal CL that was grown in soil by seed germination.

CL will be the host of Japanese encephalitis virus expression for the developmental swine vaccine.

Yellow fever virus (YFV) is a dangerous re-emerging mosquito borne flavivirus with a high lethality, which causes untreatable haemorrhagic fever in humans. Important disadvantages regarding safety and limited vaccine supply have been identified in the existing live attenuated vaccine. Alternative safer therapeutic strategies are needed to reduce the lethality of infections and protect a wider group of people. To this point, we created an array of essential tools for the study of YFV. We designed a novel YFV reporter virus to develop a highly sensitive high throughput neutralisation assay (Z’=0.65), which can screen virus inhibitors in only 48 hours. Moreover, we designed a site-specific biotinylated soluble YF envelope (E) protein by a cloning strategy involving the use of a biotin acceptor peptide and E. coli Biotin-protein birA ligase. The biotinylated E protein was used to develop a highly sensitive ELISA to screen antibodies that bind to the YFV E protein. Furthermore, we immunised mice with a soluble YFV E protein and generated anti-YFV E IgG secreting hybridoma cell lines. Monoclonal antibodies secreted by hybridomas were purified by affinity chromatography and are being characterised. The developed antibodies against YF E protein are a crucial tool for the molecular study of YF. Additionally, neutralising antibodies against YFV could potentially be developed into the first therapeutic treatment against YF infection. The toolkit developed in this project includes a neutralisation assay, a binding ELISA, and anti YFV E antibodies. These are essential instruments to expand the knowledge of YFV.

Yellow fever virus (YFV) is a dangerous re-emerging mosquito borne flavivirus with a high lethality, which causes untreatable haemorrhagic fever in humans. Important disadvantages regarding safety and limited vaccine supply have been identified in the existing live attenuated vaccine. Alternative safer therapeutic strategies are needed to reduce the lethality of infections and protect a wider group of people. To this point, we created an array of essential tools for the study of YFV. We designed a novel YFV reporter virus to develop a highly sensitive high throughput neutralisation assay (Z’=0.65), which can screen virus inhibitors in only 48 hours. Moreover, we designed a site-specific biotinylated soluble YF envelope (E) protein by a cloning strategy involving the use of a biotin acceptor peptide and E. coli Biotin-protein birA ligase. The biotinylated E protein was used to develop a highly sensitive ELISA to screen antibodies that bind to the YFV E protein. Furthermore, we immunised mice with a soluble YFV E protein and generated anti-YFV E IgG secreting hybridoma cell lines. Monoclonal antibodies secreted by hybridomas were purified by affinity chromatography and are being characterised. The developed antibodies against YF E protein are a crucial tool for the molecular study of YF. Additionally, neutralising antibodies against YFV could potentially be developed into the first therapeutic treatment against YF infection. The toolkit developed in this project includes a neutralisation assay, a binding ELISA, and anti YFV E antibodies. These are essential instruments to expand the knowledge of YFV.

The Tick Cell Biobank is the world’s only dedicated culture collection for cell lines derived from ticks and other arthropod vectors. As well as storing and distributing arthropod cell lines and training in their maintenance to UK and international researchers, the Tick Cell Biobank generates novel cell lines from arthropod species and geographic strains not already represented in the collection. Currently, efforts are focussed on European Argas, Dermacentor, Hyalomma, Rhipicephalus and Ixodes spp. ticks, Lutzomyia and Phlebotomus spp. sandflies, Culicoides spp. biting midges, Rhodnius prolixus kissing bugs and Glossina morsitans tsetse flies. Techniques used previously for ticks and insects are applied or adapted for use with embryonic or larval arthropods to generate primary cell and tissue cultures; these primary cultures are then maintained until significant cell multiplication commences and subculture can be attempted, which may take several years. This approach has yielded new cell lines from the soft tick Argas reflexus, the hard tick Hyalomma lusitanicum, the New World sand fly Lutzomyia longipalpis and the UK midge Culicoides nubeculosus. Most of these novel cell lines are now available for arbovirus research through the Tick Cell Biobank.

Flaviviruses, such as Dengue (DENV), Zika and Yellow Fever are pathogens with high morbidity and mortality. Around 390 million people per year are infected with DENV, and almost 90 million develop the clinical forms of the infection. In the present work, we analyzed the role of Aurora Kinase B (AurKB) in the replicative cycle of DENV. This Kinase regulates the activation of ESCRT-III complex, which has an essential role in the viral morphogenesis and/or budding from RE to Golgi apparatus. The compound ZM 447439 (ZM) was used to inhibit specifically AurKB, and the viral progeny, viral RNA/protein synthesis efficiency and NS1 secretion were evaluated. The kinase inhibition did not alter the viral protein production/secretion or genome replication but impaired the viral yield without altering the percentage of infected cells.

Moreover, confocal microscopy analysis of DENV-infected ZM447439-treated cells shows a delocalization of viral components from the replicative complexes. In summary, these observations indicate that AurKB participates in DENV viral morphogenesis or release. Together, our results suggest possible participation of AurKB in the viral release of budding through activation of the ESCRT-III complex and suggest a new role for AurKB on flavivirus viral cycle.

Flaviviruses, such as Dengue (DENV), Zika and Yellow Fever are pathogens with high morbidity and mortality. Around 390 million people per year are infected with DENV, and almost 90 million develop the clinical forms of the infection. In the present work, we analyzed the role of Aurora Kinase B (AurKB) in the replicative cycle of DENV. This Kinase regulates the activation of ESCRT-III complex, which has an essential role in the viral morphogenesis and/or budding from RE to Golgi apparatus. The compound ZM 447439 (ZM) was used to inhibit specifically AurKB, and the viral progeny, viral RNA/protein synthesis efficiency and NS1 secretion were evaluated. The kinase inhibition did not alter the viral protein production/secretion or genome replication but impaired the viral yield without altering the percentage of infected cells.

Moreover, confocal microscopy analysis of DENV-infected ZM447439-treated cells shows a delocalization of viral components from the replicative complexes. In summary, these observations indicate that AurKB participates in DENV viral morphogenesis or release. Together, our results suggest possible participation of AurKB in the viral release of budding through activation of the ESCRT-III complex and suggest a new role for AurKB on flavivirus viral cycle.

Mosquitoes are vectors for epidemic transmission of viruses of public and veterinary health. The mosquito vector is generally infected for life although, unlike the vertebrate counterpart, does not suffer a high fitness cost. Having a vector infected for the length of its life enables routes of viral transmission other than the classic infected bite (horizontal), including sexual (horizontal) and to the progeny (vertical). Vertical transmission is considered a route of transmission that allows for the persistence of the virus during adverse environmental periods (e.g., droughts, cold periods). Because Aedes aegypti and Aedes albopictus eggs are resistant to dessication, it is hypothesised that this attribute could promote arbovirus survival between transmission cycles, playing an important role in maintaining the pathogen.

Vertical transmission of arboviruses has been extensively documented for flaviviruses and bunyaviruses. However, there is very little and contradictory reports of vertical transmission of alphaviruses. In this research we establish the mechanisms of vertical transmission of the alphaviruses Semiliki Forest virus (SFV) and Ross river virus (RRV) and its implications in pathogen transmission of future generations.

Aedes aegypti mosquitoes were infected with SFV virus in different gonotrophic cycles. Their offspring was then reared and challenged with SFV, RRV and dengue virus (DENV). Offspring from infected parents showed significant reduction in viral load if infected with SFV or RRV but not with DENV.

Findings of this research highlight the importance of vertical transmission of alphaviruses in the general arbovirus infectious cycle.

Mosquitoes are vectors for epidemic transmission of viruses of public and veterinary health. The mosquito vector is generally infected for life although, unlike the vertebrate counterpart, does not suffer a high fitness cost. Having a vector infected for the length of its life enables routes of viral transmission other than the classic infected bite (horizontal), including sexual (horizontal) and to the progeny (vertical). Vertical transmission is considered a route of transmission that allows for the persistence of the virus during adverse environmental periods (e.g., droughts, cold periods). Because Aedes aegypti and Aedes albopictus eggs are resistant to dessication, it is hypothesised that this attribute could promote arbovirus survival between transmission cycles, playing an important role in maintaining the pathogen.

Vertical transmission of arboviruses has been extensively documented for flaviviruses and bunyaviruses. However, there is very little and contradictory reports of vertical transmission of alphaviruses. In this research we establish the mechanisms of vertical transmission of the alphaviruses Semiliki Forest virus (SFV) and Ross river virus (RRV) and its implications in pathogen transmission of future generations.

Aedes aegypti mosquitoes were infected with SFV virus in different gonotrophic cycles. Their offspring was then reared and challenged with SFV, RRV and dengue virus (DENV). Offspring from infected parents showed significant reduction in viral load if infected with SFV or RRV but not with DENV.

Findings of this research highlight the importance of vertical transmission of alphaviruses in the general arbovirus infectious cycle.

Background: Mosquito salivary glands play crucial role in transmission of arboviral diseases like Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV). We aimed to characterize virus responsive gene expression in Aedes aegypti salivary glands against these pathogenic arboviruses.

Methods: We performed high throughput RNA-sequencing on uninfected and virus-infected (DENV, ZIKV, CHIKV) female Ae. aegypti salivary glands to elucidate differential expression of genes at the transcript level. We validated the transcriptomic analysis by qPCR and performed RNA-i based functional characterization of virus-induced immune genes.

Results: DENV, ZIKV or CHIKV infected salivary gland transcriptome revealed regulations of genes related to blood feeding, metabolism, apoptosis, and immunity; the latter including Toll, IMD, and JNK pathway components. Silencing of Toll and IMD pathway components did not increase replication of all three viruses. However, depletion of infection induced JNK pathway activator and repressor showed conserved antiviral response of this pathway against the viral infections. We further showed that JNK activation by arboviruses is mediated by antiviral complement and apoptosis activation.

Conclusion: This study determined the previously unknown antiviral mechanism of JNK pathway in mosquito salivary glands against important pathogenic arboviruses. This pathway shows potential to be utilized for developing effective transmission blocking tools.