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Dengue virus (DENV) non-structural protein 5 (NS5) is critical for viral RNA synthesis within endoplasmic reticulum (ER)-derived replication complexes in the cytoplasm; however a proportion of NS5 is known to be localized to the nucleus of infected cells. The importance of nuclear DENV NS5 on viral replication and pathogenesis is still unclear. We recently discovered a nuclear localization signal (NLS) residing in the C-terminal 18 amino acid (Cter18) region of DENV NS5 and that a single NS5 P884T amino acid substitution adjacent to the NLS is sufficient to relocalize a significant proportion of DENV2 NS5 from the nucleus to the cytoplasm of infected cells. Here, in vitro studies show that the DENV2 NS5 P884T mutant replicates similarly to the parental wild-type infectious clone-derived virus while inducing a greater type I interferon and inflammatory cytokine response, in a manner independent of NS5's ability to degrade STAT2 or regulate SAT1 splicing. In both AG129 mouse and Aedes aegypti mosquito infection models, the P884T virus exhibits lower levels of viral replication only at early timepoints. Intriguingly, there appears to be a tendency for selection pressure to revert to the wild-type proline in P884T-infected Ae. aegypti, in agreement with the high conservation of the proline at this position of NS5 in DENV2, 3, and 4. These results suggest that the predominant nuclear localization of DENV NS5, while not required for viral RNA replication, may play a role in pathogenesis and modulation of the host immune response and contribute to viral fitness in the mosquito host.

Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry – a technique initially developed in mammalian cells – to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3’UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3’UTR, whereas AeStaufen interacts with both 3’UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen’s role in DENV transmission. By characterizing the first RBPs that bind to DENV2 3’UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.Author abstractDengue viruses are important human pathogens transmitted by mosquitoes. Currently, there are no effective control measures for dengue. The RNA-binding proteins (RBPs) in mosquitoes, which bind to the dengue virus genome to regulate viral multiplication, could serve as new targets for developing therapeutic interventions. In this study, we pioneered the use of RNA-affinity chromatography – a technique that identifies proteins binding to specific RNA sequences – in mosquito cells. This led to the detection of fourteen RBPs that bind to the 3’UTR of dengue virus serotype 2. We validated our results using immunoprecipitation method. Furthermore, we demonstrated that 6 of the 14 RBPs influence viral multiplication in mosquitoes. Among these six RBPs, we showed that the AePur mosquito and human homologs share an affinity to dengue virus RNA, whereas the anti-viral function is specific to the mosquito homolog. Importantly, we revealed that AeStaufen mediates a reduction of genomic and subgenomic flaviviral RNAs in multiple mosquito tissues. We also showed that the reduction of subgenomic flaviviral RNA in salivary glands diminishes the secretion of salivary subgenomic RNA, which facilitates infection at the bite site, thereby unveiling the function of AeStaufen in the virus transmission. By characterizing the first mosquito RBPs that bind to dengue virus genome, our study paves the way for leveraging these proteins as potential targets to block virus transmission.

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targeted approach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantage of DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial, to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed mutagenesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) protein that reduces 16681 infection and dissemination in both Aedes aegypti, as well as in mammalian cells to produce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a known host factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 and thus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights on DENV attenuation on a clinically tested strain.

Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3’ UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3’UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3’UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3’UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts., Author summary Dengue is a re-emerging global disease transmitted from human-to-human by mosquitoes. While environmental and host immune factors are important, viral determinants of mosquito transmission also shape the epidemiology of dengue. Understanding how dengue viruses influence transmission will help identify isolates with high epidemic potential and untangle the evolutionary pressures at play in the dual-host cycle. Here, we identified 2 substitutions in the 3’UTR of epidemic isolates that increase transmission through immune suppression in the salivary glands. Using oral infection of Aedes aegypti mosquitoes, we reported that epidemic isolates produced more subgenomic flaviviral RNA (sfRNA) in salivary glands. SfRNA is generated from the 3’UTR sequence remaining after partial genome degradation by a host nuclease. Using reverse genetics, we identified the two 3’UTR substitutions responsible for the higher sfRNA quantity in salivary glands. We further showed that these substitutions increased dengue virus titer in salivary glands and rate of saliva infection, and suppressed the Toll immune response in salivary glands. Our study identifies the substitutions that determine virus epidemiological fitness and provides a mechanism for sfRNA-mediated enhancement of transmission. Together with previous work demonstrating that sfRNA sequence modification influences dengue virus pathogenicity in human, and that shows variation in sfRNA sequence when the viruses transition from one host to vector and vice versa, our study supports that sfRNA evolution is constrained in the two hosts.

This research came from a grant from the National Research Foundation Singapore under its NIG scheme (NMRC/BNIG/2032/2015) awarded to J.P. and from the Duke-NUS Signature Research Programme funded by the Agency for Science, Technology and Research (A*STAR), Singapore, and the Ministry of Health, Singapore. Brazilian Ministry of Health, IEC and grants received from CAPES (Zika fast track grant; Coordena??o de Apoio aos Profissionais de N?vel Superior do Minist?rio da Educa??o ? Coordination of Professionals of High Level Degree of Ministry of Education); CNPq (grants: 301641/2010-2,303999/2016-0, 439971/2016-0 and 440405/2016-5; Conselho Nacional para o Desenvolvimento Cient?fico e Tecnol?gico - the Brazilian Agency for Scientific and Technological Deveelopment), and FINEP (Zika call; Financiadora de Estudos e Projetos do Minist?rio da Ci?ncia, Tecnologia e Inova??o? Agency for Financial support of Studies and Projects of the Ministry of Science, Technology and Innovation). Duke-NUS Medical School. Programme in Emerging Infectious Diseases. Singapore, Singapore / IRD. MIVEGEC UMR IRD 224-CNRS 5290-UM. Montpellier, France. Mahidol University. Faculty of Tropical Medicine. Department of Medical Entomology. Bangkok, Thailand. Duke-NUS Medical School. Programme in Emerging Infectious Diseases. Singapore, Singapore National Environment Agency. Environment Health Institute. Singapore, Singapore. IRD-Universit? de Toulouse. UMR 152 Pharma-DEV. Toulouse, France. Duke-NUS Medical School. Programme in Emerging Infectious Diseases. Singapore, Singapore Duke-NUS Medical School. Programme in Emerging Infectious Diseases. Singapore, Singapore Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. National Environment Agency. Environment Health Institute. Singapore, Singapore. IRD. MIVEGEC UMR IRD 224-CNRS 5290-UM. Montpellier, France. 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 103pfu/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.

Background Dengue and chikungunya are global re-emerging mosquito-borne diseases. In Singapore, sustained vector control coupled with household improvements reduced domestic mosquito populations for the past 45 years, particularly the primary vector Aedes aegypti. However, while disease incidence was low for the first 30 years following vector control implementation, outbreaks have re-emerged in the past 15 years. Epidemiological observations point to the importance of peridomestic infection in areas not targeted by control programs. We investigated the role of vectors in peri-domestic areas. Methods We carried out entomological surveys to identify the Aedes species present in vegetated sites in highly populated areas and determine whether mosquitoes were present in open-air areas frequented by people. We compared vector competence of Aedes albopictus and Aedes malayensis with Ae. aegypti after oral infection with sympatric dengue serotype 2 and chikungunya viruses. Mosquito saliva was tested for the presence of infectious virus particles as a surrogate for transmission following oral infection. Results We identified Aedes albopictus and Aedes malayensis throughout Singapore and quantified their presence in forested and opened grassy areas. Both Ae. albopictus and Ae. malayensis can occupy sylvatic niches and were highly susceptible to both arboviruses. A majority of saliva of infected Ae. malayensis contained infectious particles for both viruses. Conclusions Our study reveals the prevalence of competent vectors in peri-domestic areas, including Ae. malayensis for which we established the vector status. Epidemics can be driven by infection foci, which are epidemiologically enhanced in the context of low herd immunity, selective pressure on arbovirus transmission and the presence of infectious asymptomatic persons, all these conditions being present in Singapore. Learning from Singapore’s vector control success that reduced domestic vector populations, but has not sustainably reduced arboviral incidence, we suggest including peri-domestic vectors in the scope of vector management., Author summary Dengue and chikungunya are mosquito-borne diseases and re-emerging as a global burden of the 21st century. Because of the absence of cure and limitations of the current vaccine, vector control remains the sole efficient intervention to mitigate epidemics. The highly-populated city of Singapore represents an example of successful vector control, where the primary vector Aedes aegypti has practically disappeared from households. However, there as well, dengue and chikungunya are re-emerging. In this study, we surveyed peridomestic sites for mosquito vectors and identified Aedes albopictus and Aedes malayensis throughout Singapore and in open-air areas frequented by people. We further showed that both mosquito species are highly susceptible to dengue and chikungunya viruses and detected infectious virus particles in mosquito saliva, indicating their transmission capacity. Our study provides evidence that Ae. albopictus and Ae. malayensis possess all the traits necessary to contribute to virus transmission in cities and suggest that peridomestic areas be included in vector management programme.