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Summary: Understanding the transmission routes of arboviruses is key to determining their epidemiology. Here, we tested whether West Nile viruses (WNVs) are transmitted through mosquito excreta. First, we observed a high concentration of infectious units per excreta, although viruses were short lived. Second, we showed that virion excretion starts early after oral infection and remains constant for a long period, regardless of mosquito infection level. These results highlight the infectiousness of excreta from infected mosquitoes. Third, we found that both larvae and pupae were susceptible to infection, while pupae were highly permissive. Fourth, we established the proof-of-concept that immature mosquitoes can be infected by infectious excreta, demonstrating an excreta-mediated mode of transmission. Finally, by mathematically modeling excreta-mediated transmission in the field, we demonstrated that WNV can be transmitted within mosquito populations. Our study uncovers a route of transmission for mosquito-borne arboviruses, unveiling mechanisms of viral maintenance in mosquito reservoirs.

Abstract Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus with a mortality rate of up to 30%. First identified in China in 2009, it was later reported in other Asian countries, including Thailand in 2020. SFTSV has been detected in several tick species, including Rhipicephalus sanguineus, known for infesting dogs. We conducted a seroprevalence study of SFTSV in Bangkok and Nong Khai, Thailand, by analyzing 1162 human samples collected between 2019 and 2023. The testing method relied on IgG detection using ELISA and confirmed though a virus seroneutralization test. The results indicated that out of the participants, 12 (1.1%) tested positive for anti-SFTSV IgG antibodies; however, none exhibited positive results in the seroneutralization assay. Additionally, molecular detection of SFTSV, Crimean-Congo hemorrhagic fever (CCHF), Coxiella spp., Bartonella spp., and Rickettsia spp. was performed on 433 Rh. sanguineus ticks collected from 49 dogs in 2023 in Chachoengsao Province, Thailand. No evidence of these pathogens was found in ticks. These findings highlight the importance of exploring viral cross-reactivity. Furthermore, it is important to conduct additional studies to isolate SFTSV from animals and ticks in order to identify the potential transmission routes contributing to human and animal infections in Thailand.

Abstract Dengue (DENV) and chikungunya (CHIKV) viruses are among the most preponderant arboviruses. Although primarily transmitted through the bite of Aedes aegypti mosquitoes, Aedes albopictus and Aedes malayensis are competent vectors and have an impact on arbovirus epidemiology. Here, to fill the gap in our understanding of the molecular interactions between secondary vectors and arboviruses, we used transcriptomics to profile the whole-genome responses of A. albopictus to CHIKV and of A. malayensis to CHIKV and DENV at 1 and 4 days post-infection (dpi) in midguts. In A. albopictus, 1793 and 339 genes were significantly regulated by CHIKV at 1 and 4 dpi, respectively. In A. malayensis, 943 and 222 genes upon CHIKV infection, and 74 and 69 genes upon DENV infection were significantly regulated at 1 and 4 dpi, respectively. We reported 81 genes that were consistently differentially regulated in all the CHIKV-infected conditions, identifying a CHIKV-induced signature. We identified expressed immune genes in both mosquito species, using a de novo assembled midgut transcriptome for A. malayensis, and described the immune architectures. We found the JNK pathway activated in all conditions, generalizing its antiviral function to Aedines. Our comprehensive study provides insight into arbovirus transmission by multiple Aedes vectors.

Vector-borne pathogens are known to elicit immune response upon mosquito biting the host which is critically linked to the establishment of infection and pathogenesis of disease, yet the impact of the mosquito bite itself is not immunologically well-appreciated. Here the authors characterize the immune response to the mosquito vector Aedes aegypti in a controlled human in vivo challenge model and show the induction of a range of immune pathways and cell types.

Abstract Arboviruses such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses infect close to half a billion people per year, and are primarily transmitted through Aedes aegypti bites. Infection-induced changes in mosquito salivary glands (SG) influence transmission by inducing antiviral immunity, which restricts virus replication in the vector, and by altering saliva composition, which influences skin infection. Here, we profiled SG proteome responses to DENV serotype 2 (DENV2), ZIKV and CHIKV infections by using high-resolution isobaric-tagged quantitative proteomics. We identified 218 proteins with putative functions in immunity, blood-feeding or related to the cellular machinery. We observed that 58, 27 and 29 proteins were regulated by DENV2, ZIKV and CHIKV infections, respectively. While the regulation patterns were mostly virus-specific, we separately depleted four uncharacterized proteins that were upregulated by all three viral infections to determine their effects on these viral infections. Our study suggests that gamma-interferon responsive lysosomal thiol-like (GILT-like) has an anti-ZIKV effect, adenosine deaminase (ADA) has an anti-CHIKV effect, salivary gland surface protein 1 (SGS1) has a pro-ZIKV effect and salivary gland broad-spectrum antiviral protein (SGBAP) has an antiviral effect against all three viruses. The comprehensive description of SG responses to three global pathogenic viruses and the identification of new restriction factors improves our understanding of the molecular mechanisms influencing transmission.

Summary: Dengue symptoms include alteration of blood coagulation and fibrinolysis, causing severe hemorrhage and death. Here, we demonstrate that higher concentration of plasmin, the human fibrinolytic factor, in blood meal enhances dengue virus (DENV) infection in mosquito midgut and dissemination in mosquitoes. We also show that mosquitoes express a plasmin-selective Kazal-type inhibitor (AaTI) in the midgut to inhibit plasmin proteolysis and revert the enhanced infection. Using bio-layer interferometry, we show that DENV, plasmin, and AaTI interact to form a tripartite complex. Eventually, plasmin increases midgut internalization of dextran molecules and this is reverted by AaTI. Our study demonstrates that (1) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glycocalyx and enhancing DENV internalization and (2) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes. : Virology; Entomology Subject Areas: Virology, Entomology

Abstract Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.

Abstract Zika is a mosquito-borne disease associated with neurological disorders that causes an on-going pandemic. The first outbreak was recorded in Micronesia in 2007, then in French Polynesia in 2014 from which it spread to South America in 2015 and ignited a widespread epidemic. Interestingly, Zika outbreaks in Asia remained of moderate intensity although the virus is circulating. To understand these epidemiological variations, we investigated the entomological determinants of ZIKV transmission in Asia. We used oral infection of mosquitoes collected in Singapore to identify the vector species, to quantify the blood infection threshold and to compare transmissibility between an Asian ZIKV strain (H/PF13) and an American strain collected in Brazil (BE H 815744). We have confirmed the vector status of Aedes aegypti and determined that 103 pfu/ml of blood is sufficient to infect mosquitoes. We showed that only the American strain was present in the saliva 3 days post-infection, and that this strain had a 30–40% higher rate of saliva infection in Ae. aegypti from 3 to 14 days post-infection than the Asian strain. Our data suggests that American strains are more efficiently transmitted than Asian strains, which raises concerns about the introduction of American strains in Asia.

Dengue fever is caused by dengue viruses (DENV) from the Flavivirus genus and is the most prevalent arboviral disease. DENV exists in four immunogenically distinct and genetically-related serotypes (DENV-1 to 4), each subdivided in genotypes. Despite the endemicity of all four DENV serotypes in Thailand, no prior study has characterized the circulation of DENV in the southern provinces of the country. To determine the genetic diversity of DENV circulating in Southern Thailand in 2015 and 2016, we investigated 46 viruses from 182 patients' sera confirmed positive for DENV by serological and Nested RT-PCR tests. Our dataset included 2 DENV-1, 20 DENV-2, 9 DENV-3 and 15 DENV-4. Phylogenetic analysis was performed on viral envelop sequences. This revealed that part of the identified genotypes from DENV-1 and DENV-4 had been predominant in Asia (genotype I for both serotypes), while genotype II for DENV-4 and the Cosmopolitan genotype DENV-2 were also circulating. Whereas DENV-3 genotype II had been predominantly detected in South East Asia during the previous decades, we found genotype III and genotype I in Southern Thailand. All DENV genotype identified in this study were closely related to contemporary strains circulating in Southeast Asian countries, emphasizing the regional circulation of DENV. These results provide new insights into the co-circulation of all four DENV serotypes in Southern Thailand, confirming the hyperendemicity of DENV in the region. These findings also suggest a new trend of dissemination for some DENV serotypes with a possible shift in genotype distribution; as recently observed in other Asian countries.

Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although infection with ZIKV generally leads to mild disease, its recent emergence in the Americas has been associated with an increase in the development of the Guillain-Barré syndrome in adults, as well as with neurological complications, in particular congenital microcephaly, in new-borns. To date, little information is available on neuroinflammation induced by ZIKV, notably in microglial cells in the context of their metabolic activity, a series of chemical transformations that are essential for their growth, reproduction, structural maintenance and environmental responses. Therefore, in the present study we investigated the metabolomic profile of ZIKV-infected microglia. Microglial cells were exposed to ZIKV at different time points and were analyzed by a Liquid Chromatography-High Resolution mass spectrometry-based metabolomic approach. The results show that ZIKV infection in microglia leads to modulation of the expression of numerous metabolites, including lysophospholipids, particulary Lysophosphatidylcholine, and phospholipids such as Phosphatidylcholine, Phosphatidylserine, Ceramide and Sphingomyelin, and carboxylicic acids as Undecanedioic and Dodecanedioic acid. Some of these metabolites are involved in neuronal differentiation, regulation of apoptosis, virion architecture and viral replication. ZIKV infection was associated with concomitant secretion of inflammatory mediators linked with central nervous system inflammation such as IL-6, TNF-α, IL-1β, iNOS and NO. It also resulted in the upregulation of the expression of the gene encoding CX3CR1, a chemokine receptor known to regulate functional synapse plasticity and signaling between microglial cells. These findings highlight an important role for microglia and their metabolites in the process of neuroinflammation that occurs during ZIKV pathogenesis.

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.