Amino Acid Polymorphisms on the Brazilian Strain of Yellow Fever Virus Methyltransferase Are Related to the Host's Immune Evasion Mediated by Type I Interferon.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

An overview on small molecules acting as broad-spectrum agents for yellow fever infection.

INTRODUCTION: Yellow fever virus (YFV) is a mosquito-borne flavivirus, endemic in 47 countries in Africa and South America, which causes febrile symptoms that can evolve in 15% of the patients to serious hemorrhagic conditions, liver injury, and multiorgan failure. Although a highly effective vaccine (YF-17D vaccine) is available, to date, no antiviral drugs have been approved for the prevention and treatment of YFV infections. AREAS COVERED: This review article focuses on the description of viral targets that have been considered within YFV and flavivirus drug discovery studies and on the most relevant candidates reported so far that elicit broad-spectrum inhibition against relevant strains and mutants of YFV. EXPERT OPINION: Considering the growing interest on (re)emerging vector-borne viral infections, it is expected that flavivirus drug discovery will quickly deliver potential candidates for clinical evaluation. Due to similarity among flaviviral targets, several candidates identified against different flaviviruses have shown broad-spectrum activity, thus exhibiting anti-YFV activity, as well. In this regard, it would be desirable to routinely include the assessment of antiviral activity against different YFV strains. On the other hand, the development of host targeting agents are still at an initial stage and deserve further focused efforts.

High-Throughput Platform for Detection of Neutralizing Antibodies Using Flavivirus Reporter Replicon Particles.

Flavivirus outbreaks require fast and reliable diagnostics that can be easily adapted to newly emerging and re-emerging flaviviruses. Due to the serological cross-reactivity among flavivirus antibodies, neutralization tests (NT) are considered the gold standard for sero-diagnostics. Here, we first established wild-type single-round infectious virus replicon particles (VRPs) by packaging a yellow fever virus (YFV) replicon expressing Gaussia luciferase (Gluc) with YFV structural proteins in trans using a double subgenomic Sindbis virus (SINV) replicon. The latter expressed the YFV envelope proteins prME via the first SINV subgenomic promoter and the capsid protein via a second subgenomic SINV promoter. VRPs were produced upon co-electroporation of replicon and packaging RNA. Introduction of single restriction enzyme sites in the packaging construct flanking the prME sequence easily allowed to exchange the prME moiety resulting in chimeric VRPs that have the surface proteins of other flaviviruses including dengue virus 1--4, Zika virus, West Nile virus, and tick-borne encephalitis virus. Besides comparing the YF-VRP based NT assay to a YF reporter virus NT assay, we analyzed the neutralization efficiencies of different human anti-flavivirus sera or a monoclonal antibody against all established VRPs. The assays were performed in a 96-well high-throughput format setting with Gluc as readout in comparison to classical plaque reduction NTs indicating that the VRP-based NT assays are suitable for high-throughput analyses of neutralizing flavivirus antibodies.

Mapping environmental suitability of Haemagogus and Sabethes spp. mosquitoes to understand sylvatic transmission risk of yellow fever virus in Brazil.

BACKGROUND: Yellow fever (YF) is an arboviral disease which is endemic to Brazil due to a sylvatic transmission cycle maintained by infected mosquito vectors, non-human primate (NHP) hosts, and humans. Despite the existence of an effective vaccine, recent sporadic YF epidemics have underscored concerns about sylvatic vector surveillance, as very little is known about their spatial distribution. Here, we model and map the environmental suitability of YF's main vectors in Brazil, Haemagogus spp. and Sabethes spp., and use human population and NHP data to identify locations prone to transmission and spillover risk. METHODOLOGY/PRINCIPAL FINDINGS: We compiled a comprehensive set of occurrence records on Hg. janthinomys, Hg. leucocelaenus, and Sabethes spp. from 1991-2019 using primary and secondary data sources. Linking these data with selected environmental and land-cover variables, we adopted a stacked regression ensemble modelling approach (elastic-net regularized GLM, extreme gradient boosted regression trees, and random forest) to predict the environmental suitability of these species across Brazil at a 1 km x 1 km resolution. We show that while suitability for each species varies spatially, high suitability for all species was predicted in the Southeastern region where recent outbreaks have occurred. By integrating data on NHP host reservoirs and human populations, our risk maps further highlight municipalities within the region that are prone to transmission and spillover. CONCLUSIONS/SIGNIFICANCE: Our maps of sylvatic vector suitability can help elucidate potential locations of sylvatic reservoirs and be used as a tool to help mitigate risk of future YF outbreaks and assist in vector surveillance. Furthermore, at-risk regions identified from our work could help disease control and elucidate gaps in vaccination coverage and NHP host surveillance.

The host range restriction of bat-associated no-known-vector flaviviruses occurs post-entry.

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.

Inhibition of Orbivirus Replication by Fluvastatin and Identification of the Key Elements of the Mevalonate Pathway Involved.

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus), dengue virus (Flavivirus), African swine fever virus (Asfarviridae) and poliovirus (Picornaviridae). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR(-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Yellow Fever Outbreak in Eastern Senegal, 2020-2021.

Yellow fever virus remains a major threat in low resource countries in South America and Africa despite the existence of an effective vaccine. In Senegal and particularly in the eastern part of the country, periodic sylvatic circulation has been demonstrated with varying degrees of impact on populations in perpetual renewal. We report an outbreak that occurred from October 2020 to February 2021 in eastern Senegal, notified and managed through the synergistic effort yellow fever national surveillance implemented by the Senegalese Ministry of Health in collaboration with the World Health Organization, the countrywide 4S network set up by the Ministry of Health, the Institut Pasteur de Dakar, and the surveillance of arboviruses and hemorrhagic fever viruses in human and vector populations implemented since mid 2020 in eastern Senegal. Virological analyses highlighted the implication of sylvatic mosquito species in virus transmission. Genomic analysis showed a close relationship between the circulating strain in eastern Senegal, 2020, and another one from the West African lineage previously detected and sequenced two years ago from an unvaccinated Dutch traveler who visited the Gambia and Senegal before developing signs after returning to Europe. Moreover, genome analysis identified a 6-nucleotide deletion in the variable domain of the 3'UTR with potential impact on the biology of the viral strain that merits further investigations. Integrated surveillance of yellow fever virus but also of other arboviruses of public health interest is crucial in an ecosystem such as eastern Senegal.

Incidence, drivers and global health implications of the 2019/2020 yellow fever sporadic outbreaks in Sub-Saharan Africa.

The 2019 and 2020 sporadic outbreaks of yellow fever (YF) in Sub-Saharan African countries had raised a lot of global health concerns. This article aims to narratively review the vector biology, YF vaccination program, environmental factors and climatic changes, and to understand how they could facilitate the reemergence of YF. This study comprehensively reviewed articles that focused on the interplay and complexity of YF virus (YFV) vector diversity/competence, YF vaccine immunodynamics and climatic change impacts on YFV transmission as they influence the 2019/2020 sporadic outbreaks in Sub-Saharan Africa (SSA). Based on available reports, vectorial migration, climatic changes and YF immunization level could be reasons for the re-mergence of YF at the community and national levels. Essentially, the drivers of YFV infection due to spillover are moderately constant. However, changes in land use and landscape have been shown to influence sylvan-to-urban spillover. Furthermore, increased precipitation and warmer temperatures due to climate change are likely to broaden the range of mosquitoes' habitat. The 2019/2020 YF outbreaks in SSA is basically a result of inadequate vaccination campaigns, YF surveillance and vector control. Consequently, and most importantly, adequate immunization coverage must be implemented and properly achieved under the responsibility of the public health stakeholders.

Yellow fever in Asia-a risk analysis.

BACKGROUND: There is concern about the risk of yellow fever (YF) establishment in Asia, owing to rising numbers of urban outbreaks in endemic countries and globalisation. Following an outbreak in Angola in 2016, YF cases were introduced into China. Prior to this, YF had never been recorded in Asia, despite climatic suitability and the presence of mosquitoes. An outbreak in Asia could result in widespread fatalities and huge economic impact. Therefore, quantifying the potential risk of YF outbreaks in Asia is a public health priority. METHODS: Using international flight data and YF incidence estimates from 2016, we quantified the risk of YF introduction via air travel into Asia. In locations with evidence of a competent mosquito population, the potential for autochthonous YF transmission was estimated using a temperature-dependent model of the reproduction number and a branching process model assuming a negative binomial distribution. RESULTS: In total, 25 cities across Asia were estimated to be at risk of receiving at least one YF viraemic traveller during 2016. At their average temperatures, we estimated the probability of autochthonous transmission to be <50% in all cities, which was primarily due to the limited number of estimated introductions that year. CONCLUSION: Despite the rise in air travel, we found low support for travel patterns between YF endemic countries and Asia resulting in autochthonous transmission during 2016. This supports the historic absence of YF in Asia and suggests it could be due to a limited number of introductions in previous years. Future increases in travel volumes or YF incidence can increase the number of introductions and the risk of autochthonous transmission. Given the high proportion of asymptomatic or mild infections and the challenges of YF surveillance, our model can be used to estimate the introduction and outbreak risk and can provide useful information to surveillance systems.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

αVß3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells.

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVß3 integrin (MEF-αVß3-/-), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVß3 integrin absence in double-knockout MEF-αVß3-/- on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVß3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVß3 integrin expression. Most interestingly, the deletion of αVß3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVß3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.

Consumptive coagulopathy of severe yellow fever occurs independently of hepatocellular tropism and massive hepatic injury.

Yellow fever (YF) is a mosquito-transmitted viral disease that causes tens of thousands of deaths each year despite the long-standing deployment of an effective vaccine. In its most severe form, YF manifests as a hemorrhagic fever that causes severe damage to visceral organs. Although coagulopathy is a defining feature of severe YF in humans, the mechanism by which it develops remains uncertain. Hepatocytes are a major target of yellow fever virus (YFV) infection, and the coagulopathy in severe YF has long been attributed to massive hepatocyte infection and destruction that results in a defect in clotting factor synthesis. However, when we analyzed blood from Brazilian patients with severe YF, we found high concentrations of plasma D-dimer, a fibrin split product, suggestive of a concurrent consumptive process. To define the relationship between coagulopathy and hepatocellular tropism, we compared infection and disease in Fah-/-, Rag2-/-, and Il2rɣ-/- mice engrafted with human hepatocytes (hFRG mice) and rhesus macaques using a highly pathogenic African YFV strain. YFV infection of macaques and hFRG mice caused substantial hepatocyte infection, liver damage, and coagulopathy as defined by virological, clinical, and pathological criteria. However, only macaques developed a consumptive coagulopathy whereas YFV-infected hFRG mice did not. Thus, infection of cell types other than hepatocytes likely contributes to the consumptive coagulopathy associated with severe YF in primates and humans. These findings expand our understanding of viral hemorrhagic disease and associated coagulopathy and suggest directions for clinical management of severe YF cases.

Risk of yellow fever virus transmission in the Asia-Pacific region.

Historically endemic to Sub-Saharan Africa and South America, yellow fever is absent from the Asia-Pacific region. Yellow fever virus (YFV) is mainly transmitted by the anthropophilic Aedes mosquitoes whose distribution encompasses a large belt of tropical and sub tropical regions. Increasing exchanges between Africa and Asia have caused imported YFV incidents in non-endemic areas, which are threatening Asia with a new viral emergence. Here, using experimental infections of field-collected mosquitoes, we show that Asian-Pacific Aedes mosquitoes are competent vectors for YFV. We observe that Aedes aegypti populations from Singapore, Taiwan, Thailand, and New Caledonia are capable of transmitting YFV 14 days after oral infections, with a number of viral particles excreted from saliva reaching up to 23,000 viral particles. These findings represent the most comprehensive assessment of vector competence and show that Ae. aegypti mosquitoes from the Asia-Pacific region are highly competent to YFV, corroborating that vector populations are seemingly not a brake to the emergence of yellow fever in the region.

Establishment and characterization of a cell line from Ghanaian Aedes aegypti (Diptera: Culicidae) focusing on Aedes-borne flavivirus susceptibility.

Mosquitoes are generally considered one of the most important vectors of arboviruses, with Aedes aegypti regarded as the most important in transmission of yellow fever and dengue viruses. To investigate why there are differences in the incidence of dengue fever and Zika in different geographical areas and an absence of outbreaks in Ghana in spite of an abundance of A. aegypti mosquitoes, we established a continuous cell line from embryonic cells of A. aegypti collected in Ghana and assessed its susceptibility to dengue, yellow fever, and Zika viruses. The new cell line (designated AeAe-GH98), having an adhesive spindle-shaped web-like morphology, was serially subcultured in both VP-12 and Schneider's medium supplemented with 10% heat-inactivated fetal bovine serum. AeAe-GH98 cells were found to have a population doubling time of 1.3 d during exponential growth. The mosquito colony used to establish the cell line was confirmed to have originated from Africa using microsatellite assay. In terms of susceptibility to Aedes-borne flaviviruses, AeAe-GH98 cells were found to have different degrees of susceptibility to yellow fever, Zika, and dengue virus infection and propagation. While susceptibility of AeAe-GH98 cells to yellow fever and Zika viruses was comparable with that of C6/36 cells, susceptibility to dengue virus was significantly lower. This cell line will serve as a useful tool for determining molecular factors influencing virus-vector susceptibility in vitro.

Generation of a reporter yellow fever virus for high throughput antiviral assays.

Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.

Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses.

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating.

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

Confronting the Multidimensional Challenges of Research in the Context of Emerging Infectious Diseases in Brazil: The Example of Yellow Fever.

In the most recent Brazilian yellow fever (YF) outbreak, a group of clinicians and researchers initiated in mid-January 2018 a considerable effort to develop a multicenter randomized controlled clinical trial to evaluate the effect of sofosbuvir on YF viremia and clinical outcomes (Brazilian Clinical Trials Registry: RBR-93dp9n). The approval of this protocol had urgency given the seasonal/short-lived pattern of YF transmission, large number of human cases, and epidemic transmission at the outskirts of a large urban center. However, many intricacies in the research regulatory and ethical submission systems in Brazil were indomitable even under such pressing conditions. By April 2018, we had enrolled 29 patients for a target sample size of 90 participants. Had enrollment been initiated 3 weeks earlier, an additional 31 patients could have been enrolled, reaching the prespecified sample size for the interim analysis. This recent experience highlights the urgent need to improve local preparedness for research in the setting of explosive outbreaks, as has been seen in the last few years in different countries.

Potential of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) to transmit yellow fever virus in urban areas in Central Africa.

Yellow Fever (YF) remains a major public health issue in Sub-Saharan Africa and South America, despite the availability of an effective vaccine. In Africa, most YF outbreaks are reported in West Africa. However, urban outbreaks occurred in 2016 in both Angola and the Democratic Republic of Congo (DRC), and imported cases were reported in Chinese workers coming back from Africa. In Central Africa, Cameroon and the Republic of Congo host a high proportion of non-vaccinated populations increasing the risk of urban outbreaks. The main vector is Aedes aegypti and possibly, Aedes albopictus, both being anthropophilic and domestic mosquitoes. Here, we provide evidence that both Ae. aegypti and Ae. albopictus in Cameroon and the Republic of Congo are able to transmit Yellow fever virus (YFV) with higher rates of infection, dissemination, and transmission for Ae. aegypti. We conclude that the potential of both Aedes species to transmit YFV could increase the risk of urban YF transmission and urge public health authorities to intensify their efforts to control domestic vectors, and extend vaccine coverage to prevent major YFV outbreak.

A peridomestic Aedes malayensis population in Singapore can transmit yellow fever virus.

The case-fatality rate of yellow fever virus (YFV) is one of the highest among arthropod-borne viruses (arboviruses). Although historically, the Asia-Pacific region has remained free of YFV, the risk of introduction has never been higher due to the increasing influx of people from endemic regions and the recent outbreaks in Africa and South America. Singapore is a global hub for trade and tourism and therefore at high risk for YFV introduction. Effective control of the main domestic mosquito vector Aedes aegypti in Singapore has failed to prevent re-emergence of dengue, chikungunya and Zika viruses in the last two decades, raising suspicions that peridomestic mosquito species untargeted by domestic vector control measures may contribute to arbovirus transmission. Here, we provide empirical evidence that the peridomestic mosquito Aedes malayensis found in Singapore can transmit YFV. Our laboratory mosquito colony recently derived from wild Ae. malayensis in Singapore was experimentally competent for YFV to a similar level as Ae. aegypti controls. In addition, we captured Ae. malayensis females in one human-baited trap during three days of collection, providing preliminary evidence that host-vector contact may occur in field conditions. Finally, we detected Ae. malayensis eggs in traps deployed in high-rise building areas of Singapore. We conclude that Ae. malayensis is a competent vector of YFV and re-emphasize that vector control methods should be extended to target peridomestic vector species.